A strain of Streptomyces fusiformis and its application

Abstract

The present invention relates to a strain of Streptomyces netropsis and its application. The strain name is Streptomyces netropsis JN4-2, and the deposit number is CGMCC No.28157. The strain and its bacterial agent can be used for biological control of pathogenic bacteria such as red pine damping-off pathogen, red bean anthracnose pathogen, aggregated small hole shell fungus, apple shell monochrome septum spore canker pathogen, corn large spot pathogen, tobacco target spot pathogen, rice seedling 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 red star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, pear Boshi disk fruit rot pathogen, etc., and has the advantages of good biological control effect, wide antibacterial spectrum, wide application range, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Streptomyces fusiformis and an application thereof. Background Art

[0002] Korean pine (Pinus koraiensis) belongs to the genus Pinus of the family Pinaceae. It is native to the northern region of Eurasia and is the main community-building and dominant species in the northeastern forest region of my country. Korean pine is a light-loving plant with strong cold resistance and good corrosion resistance. It is suitable for planting in moist slightly acidic or neutral soil. Atmospheric humidity above 0.7 promotes the growth of Korean pine.

[0003] Korean pine damping-off is caused by fungi (mainly Rhizoctonia and Fusarium), a common and serious infectious disease of seedlings. After the seedlings are killed, the mortality rate is extremely high, and the incidence rate of 1-2 year old seedlings is extremely high, reaching more than 60% in severe cases, and even total failure. Chemical control and soil disinfectant treatment are still mainly used in production to prevent the occurrence and harm of this disease. The excessive use of chemical pesticides causes pathogens to develop drug resistance and 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 microbial communities, which further leads to poor nutritional conditions and weak growth of seedlings. There are problems such as low survival rate and poor adaptability in subsequent afforestation and ecological restoration. In the past 40 years, my country's agricultural production has relied on chemical inputs, and the use of pesticides has increased significantly. According to statistics from the National Environmental Protection Agency, in 2007, my country produced about 250,000 tons of pesticide raw materials, which were processed into more than 800,000 tons of preparations and applied to the environment through various means. 10%-20% attached to plants, and 80%-90% scattered in the soil, water and atmosphere (Yang Yonghua, Yao Jian, Hua Xiaomei. Effects of pesticide pollution on the functional diversity of soil microbial communities [J]. Journal of Microbiology, 2000, (02): 23-25+47.). In the face of the above problems, biological control is becoming more and more popular.

[0004] In the 1960s, Wu Yousan et al. first reported the disease in China. Since then, studies on the occurrence and prevention of the disease have been common. The occurrence of pine seedling damping-off 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 pine seedling damping-off in Northeast China is mainly affected by soil moisture content and rainfall. High rainfall and high soil moisture content are conducive to the disease. In addition, the longer the disease is delayed, the higher the overall incidence rate (Wu Yousan, Gao Ya, Gu Sifang et al. Research on pine seedling damping-off II. Disease prevalence [J]. Acta Phytophylacica Sinica, 1963, (04): 399-408.). Tong Ying et al. (1979) found through experiments that the pathogens causing pine seedling blight are mainly Rhizoctonia, Fusarium and Pythium, with Rhizoctonia being the most pathogenic ([4] Tong Ying. Pine seedling blight and its prevention and control [J]. Jilin Forestry Science and Technology, 1979, (02): 117-120.). Zhang Lijun et al. (2012) found that the red pine blight pathogen overwinters in the remains and soil of various hosts as hyphae and spores, and spreads between rows through hyphae. 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. The occurrence law and prevention and control technology of red pine blight [J]. Agriculture and Technology, 2012, 32 (09): 116.).

[0005] The harm caused by plant diseases to agricultural and forestry production is becoming more and more serious, and the economic losses are immeasurable. As the disadvantages of chemical control are gradually aggravated, biological control has received 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 antibiotics reported in the world, actinomycetes account for more than half. Actinomycetes are the first type of microorganisms discovered to have biological control effects. Among actinomycetes, Streptomyces is the genus that produces the most antibacterial substances. Gao Yuhong et al. (2009) from Northeast Agricultural University reported a new agricultural antibiotic, doramectin, which is an avermectin antibiotic produced by fermentation of a new species of recombinant Streptomyces avermitilis. Doramectin is currently one of the best antiparasitic drugs in the avermectin family and is a dual-acting agent that has good killing effects on nematodes and arthropods (Jiang Wei. Mutation biosynthesis of doramectin and optimization of fermentation conditions [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 an inhibition rate of 100% on the 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 suitable 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

[0006] In view of the problems existing in the prior art, the present invention provides a strain of Streptomyces fusiformis and its application. The strain is discovered for the first time by the present invention, and further verification shows that the strain has the advantages of good biocontrol effect, broad antibacterial spectrum, wide application range, etc.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The present invention 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 was deposited in the General Microbiology Center of China Culture Collection Administration (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.

[0009] The present invention obtains multiple actinomycetes by isolating from soil collected from different areas of Jilin Province, and adopts multiple screening and control effect determination, and finally obtains an antagonistic strain with the best effect of inhibiting red pine damping-off pathogen, which is marked as JN4-2. This achieves a breakthrough in the field of actinomycetes in the control of red pine damping-off disease, and provides new strains and products for the biological control of red pine damping-off disease.

[0010] The present invention provides a bacterial agent, including the above-mentioned Streptomyces fusiformis and / or the fermentation product of the above-mentioned Streptomyces fusiformis. The present invention has no special restrictions on the dosage form of the bacterial agent. For example, the bacterial 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 preparations can also be added to facilitate their application.

[0011] The Streptomyces fusiformis and the bacterial agent thereof provided by the invention have the advantages of good biocontrol effect, broad antibacterial spectrum, wide application range, and the like.

[0012] The present invention provides a fermentation method of the above-mentioned Streptomyces fusiformis, comprising the following steps: inoculating the above-mentioned Streptomyces fusiformis with a culture medium for fermentation and culturing. The formula of the fermentation culture medium may include: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate, per 1000mL of distilled water, pH 7.0. The fermentation culture conditions may include: culturing at 28°C for 6d.

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

[0014] The 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 150r / min constant temperature shaking for 6 days, then centrifuging and taking the supernatant.

[0015] The formula of the fermentation medium may include: 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.

[0016] The method for preparing the seed solution may include the following steps: streaking the above-mentioned Streptomyces fusiformis on a plate for culture, and scraping spores into sterile water to prepare a spore suspension, namely the seed solution.

[0017] The present invention provides the use of the above-mentioned Streptomyces fusiformis in the prevention and treatment of plant pathogens. The strain provided by the present invention can be used to prepare a biocontrol agent, which is used to prevent and treat one or more of the following: red pine blight pathogen, red bean anthracnose pathogen, aggregated burrowing fungus, apple shell monochromatic spore canker pathogen, corn leaf spot pathogen, tobacco target spot pathogen, rice seedling 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 spot pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen; and is used to prevent and treat diseases caused by the above-mentioned pathogens.

[0018] 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: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr fungus, apple shell monochromatic spore canker pathogen, corn leaf spot pathogen, tobacco target spot pathogen, rice seedling 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 spot pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen. 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.

[0019] The present invention provides an application of supernatant in the prevention and treatment of plant pathogenic bacteria. The supernatant can be prepared by the following method: inoculating the seed liquid of the streptomyces into a fermentation medium, culturing at 28°C and 150r / min constant temperature shaking for 6 days, then centrifuging and taking the supernatant.

[0020] The formula of the fermentation medium may include: 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.

[0021] The method for preparing the seed liquid comprises the following steps: streaking and culturing the streptomyces on a plate, taking spores and scraping them into sterile water to prepare a spore suspension, namely the seed liquid.

[0022] The supernatant provided by the present invention can be used to prevent and treat the diseases caused by the above-mentioned plant pathogenic bacteria.

[0023] The invention has the advantages of good prevention and treatment effect, broad antibacterial spectrum, wide application range, etc. when used.

[0024] 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.

[0025] 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

[0026] Figure 1 These are the experimental results of Example 1 of the present invention, wherein A is the inhibitory effect of JN4-2 live bacteria on the pathogen of Korean pine, and B is the pathogen of Korean pine.

[0027] Figure 2 These are the culture characteristics of JN4-2 on Gao's No. 1 agar medium.

[0028] Figure 3 This is the morphology of JN4-2 under optical microscope.

[0029] Figure 4 Phylogenetic analysis of strain JN4-2 and related strains.

[0030] Figure 5 These are the test results of the inhibition zone of JN4-2 live bacteria against Aspergillus niger, where A is the detection result of JN4-2 live bacteria against Aspergillus niger, and B is the control (Aspergillus niger).

[0031] Figure 6 These are the test results of the inhibition zone of JN4-2 live bacteria against apple fruit rot pathogens, where A is the JN4-2 live bacteria against apple fruit rot pathogens, and B is the control (apple fruit rot pathogens).

[0032] Figure 7 These are the test results of the inhibition zone of JN4-2 live bacteria against tobacco target spot pathogen, wherein A is the detection result of JN4-2 live bacteria against tobacco target spot pathogen, and B is the control (tobacco target spot pathogen).

[0033] Figure 8 These are the test results of the inhibition zone of JN4-2 live bacteria against Pythium aphanidermatum, among which A is the detection result of JN4-2 live bacteria against Pythium aphanidermatum, and B is the control melon (Pythium aphanidermatum control).

[0034] Fig. 9 These are the test results of the inhibition zone of JN4-2 fermentation liquid against Rhizoctonia solani, where A is the inhibition zone of JN4-2 fermentation liquid against Rhizoctonia solani, and B is the control (Rhizoctonia solani).

[0035] Fig.10 The results are as follows: JN4-2 fermentation broth against red bean anthracnose bacteria inhibition zone test results, wherein A is JN4-2 fermentation broth against red bean anthracnose bacteria, and B is the control (red bean anthracnose bacteria).

[0036] Fig.11 The results of the inhibition zone test of JN4-2 fermentation liquid on muskmelon vine disease pathogenic bacteria are shown in Figure 1, wherein A is the inhibition zone of JN4-2 fermentation liquid on muskmelon vine disease pathogenic bacteria, and B is the control (muskmelon vine disease pathogenic bacteria).

[0037] Fig.12 These are the test results of the inhibition zone of JN4-2 fermentation liquid against larch dieback pathogen, where A is the inhibition zone of JN4-2 fermentation liquid against larch dieback pathogen, and B is the control (larch dieback pathogen). DETAILED DESCRIPTION

[0038] 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.

[0039] The present invention isolated 87 actinomycetes from the understory soil of various forest areas in Jilin Province, screened with red pine damping-off bacteria as the target pathogen, and screened out the strain JN4-2 with the best inhibitory effect on red pine damping-off bacteria. The strain JN4-2 was determined to be a spindle Streptomyces by morphological observation and molecular biological analysis and identification methods such as 16SrDNA analysis. Therefore, it was named as spindle Streptomyces netropsis JN4-2 (Streptomyces netropsis JN4-2), and it was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on August 11, 2023, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 28157.

[0040] 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.

[0041] The tested plant pathogens: Fusarium oxysporum, Valsa sordida, Dothiorella gregari, Botryosphaeria stevensii, Botryosphaeria dothidea, Neofusicoccumlaricinum, Monilinia polystroma, Valsa mali, were isolated and preserved by Jilin Academy of Forestry Sciences; Colletotrichum sp, Exserohilum turcicum, Rhizoctonia solani, F. moniliforme, Fusarium oxysporum, C. gloeosporioides, Phomopsis, were isolated and preserved by Jilin Academy of Forestry Sciences; vexans, Pythium aphanidermatum, Alternaria alternata, and Sclerotinias cleroriorum were donated by the Plant Virus Research Laboratory of Shenyang Agricultural University; Botryosphaeria dothidea and Neofusicoccumparvum were donated by Associate Professor Xu Chengnan of the School of Life Sciences of Yan'an University; 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.

[0042] 26 soil samples were collected from Hunchun, Dunhua, Helong, Jiangnan Forest Farm and other places in Jilin Province.

[0043] Test medium:

[0044] Gause's No.1 agar medium: KNO31g, K2HPO40.5g, MgSO40.5g, NaCl0.5g, FeSO40.01g, soluble starch 20g, agar 20g, per 1000mL water, pH 7.2-7.4.

[0045] Potato dextrose agar (PDA): 200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water.

[0046] Fermentation medium: 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.

[0047] Test reagents:

[0048] Ezup column bacterial genomic DNA extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. (referred to as 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 Biotech (Dalian) Co., Ltd.; the others were domestically produced analytical grade.

[0049] Universal primers 27F and 1429R were purchased from Changchun Kumei Biotechnology Co., Ltd.

[0050] Instrument: BX53 Olympus optical microscope was purchased from Olympus Corporation.

[0051] In the embodiments, if data processing is involved, SPSS 23.0 is used for data statistics; the phylogenetic tree is established using MEGA X version.

[0052] 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.

[0053] The following is an introduction through specific embodiments.

[0054] Example 1 Screening of biocontrol Streptomyces strains

[0055] From June to July 2022, 26 soil samples were collected from Hunchun, Dunhua, Helong, Jilin Province, and Jiangnan Forest Farm, Panshi City, Jilin City, Jilin Province. 100 ml of sterile water was fully mixed with 10 g of soil sample and then allowed to stand to dilute to 10 -3 , 10 -4 , 10 -5 Different concentrations were spread on Gao's No. 1 agar medium to separate and screen the strains. After individual colonies grew out of the culture medium, they were transferred to Gao's No. 1 agar medium for purification culture. The purification culture was repeated 3 times. The screened strains were numbered, transferred to slant surfaces and stored in a 4°C refrigerator for later use.

[0056] After separation and purification by dilution culture method, 87 purified strains with different shapes, sizes and colors were obtained. The antibacterial activity of the above strains was detected by plate confrontation test with Rhizoctonia solani as the target bacteria.

[0057] The experimental method of the plate confrontation test includes the following steps: culturing the obtained strain until it grows vigorously, taking the red pine damping-off pathogen as the target strain, and determining the inhibitory effect of the isolated strain on the red pine damping-off pathogen. The specific method is as follows: Use a hole puncher to make a 7mm bacterial cake on the red pine damping-off pathogen plate, place the pathogenic bacteria cake on one side 2cm above and below the center of the PDA plate (diameter 90mm), and use an inoculation loop to pick the cultured test strain on one side to draw a line, and use a plate without inoculation of actinomycetes as the control group, and culture at a constant temperature of 28°C. When the colony of the control group fills the plate, use a vernier caliper to measure the average distance between the pathogenic bacteria cake and the test strain, and repeat 3 times for each group. Select the strain with vigorous growth and the best antibacterial effect as the next research object.

[0058] The results of the plate confrontation test showed that among the 87 strains screened, 14 strains showed obvious antibacterial effects (Table 1). Among them, JN4-2 collected from Jiangnan Forest Farm had the best antibacterial effect on Korean pine damping-off pathogens. The antibacterial zone of JN4-2 could reach 22.54 mm ( Figure 1 ). Therefore, JN4-2 was selected as the object for further study.

[0059] Table 1 Antibacterial activity of live Streptomyces against red pine blight pathogen

[0060]

[0061] The data in the table are mean ± standard deviation.

[0062] Example 2 Morphological observation of JN4-2 strain

[0063] The strain JN4-2 was inoculated on Gao's agar medium No. 1, and the sterilized cover slip was inserted into the medium at a 45° angle. The culture was cultured at 28°C for about 5 days, and the cover slip was taken out and placed under a BX53 Olympus optical microscope to observe the morphology of the strain. According to the International Streptomyces Project and the research methods of the Actinomycetes Classification Group of the Institute of Microbiology, Chinese Academy of Sciences, the culture medium for actinomycete identification was used to observe its growth status and appearance characteristics.

[0064] Morphological observation results of aerial hyphae and spore hyphae: strain JN4-2 grew radially on Gao's agar medium No. 1, and the aerial hyphae were very lush. When cultured at 28℃ for 1-3 days, the colonies were round and smooth, and no spores were produced. On the 5th day, white spores grew from the edge of the colony, and gradually turned into fleshy white as the number of days of growth increased. The color of the matrix mycelium was yellow, and no soluble pigment was produced. After continuous culture for 30 days, the colony turned cream yellow ( Figure 2 ). Under the microscope, it can be seen that the hyphae are numerous and branched, slender, straight, unbroken, without transverse membranes, and with a large number of spores scattered around the hyphae ( Figure 3).

[0065] Example 3 Identification of JN4-2 strain

[0066] The molecular biological identification of JN4-2 was performed by 16S rDNA sequencing, including the following steps: the genomic DNA of JN4-2 was extracted using the Ezup column-type bacterial genomic DNA extraction kit (Sangon Biotech Co., Ltd.).

[0067] 16S rDNA Bacterial Identification PCR Kit was used for PCR amplification. The PCR reaction system (50 μL) included: PCR Mixture 25 μL, Template 2 μL, universal primers 27F, 1429R 2 μL each, ddH2O 19 μL. PCR reaction conditions: 94℃ denaturation for 4 min; 94℃ denaturation for 30 s, 58℃ renaturation for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min, 4℃ storage, PCR product with sterile water as negative control, PCR product of strains with 16SrRNA results determined in the laboratory as positive control, loading volume 2 μL, 2% agarose gel electrophoresis (DNA Marker DL2000). Agarose Gel DNA Purification Kit Ver 2.0 was used to recover the PCR product. The recovered product was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing and analysis. The obtained strain 16s sequences were spliced, and the measured sequences were compared with the sequences of known similar model species downloaded from GenBank using the NCBI BLAST program. The sequences were aligned and the phylogenetic tree was drawn using the neighbor-joining method (NJ) of MEGA X software.

[0068] After PCR amplification, purification, and sequencing, the length of the 16S rDNA sequence of strain JN4-2 was measured to be 1409 bp. Twelve sequences with high homology to strain JN4-2 were selected to establish a phylogenetic tree, such as Figure 4 As shown, compared with the 16SrDNA sequence 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) were clustered in the same branch and had the closest relationship. Strain JN4-2 (OR758449) was identified as Streptomyces netropsis.

[0069] Therefore, it was named Streptomyces netropsis JN4-2, and was deposited in the General Microbiology Center of China Culture Collection (CGMCC) on August 11, 2023. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No. 28157.

[0070] Example 4 Determination of antibacterial activity of antagonistic strain JN4-2

[0071] The plate confrontation test was used to detect the antibacterial activity of the antagonistic strain JN4-2 against the test strain. The test method of the plate confrontation test was referred to Example 1.

[0072] The test strains are: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr shell fungus, apple shell monochrome 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 red star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen.

[0073] The experimental results showed that the antagonistic bacteria JN4-2 had an inhibitory effect on all the tested strains. Among them, JN4-2 had the strongest inhibitory effect on the red pine blight pathogen, with an obvious inhibition zone of 22.54 mm in width, which was significantly different from the other tested pathogens (Table 2); Figure 5 ), blueberry canker and apple fruit rot ( Figure 6 ) also had a strong inhibitory effect, with the inhibition bandwidths being 17.19mm, 16.23mm, and 16.33mm respectively, against tobacco target spot pathogen ( Figure 7 ), corn leaf blight, apigenin, and phytophthora aphaniderm ( Figure 8 ) and other inhibitory effects were relatively weak, but the width of the inhibition zone still reached more than 10.13 mm.

[0074] Table 2 Antibacterial spectrum of live bacteria of antagonistic bacteria JN4-2

[0075]

[0076] The data in the table are mean ± standard deviation.

[0077] Example 5 Preparation of fermentation broth of strain JN4-2 and determination of its antibacterial spectrum

[0078] Since strain JN4-2 has the strongest antibacterial activity, strain JN4-2 was selected for cultivation in fermentation medium. Strain JN4-2 was streaked and cultured on a plate of Gao's No. 1 agar medium. After it grew vigorously, the spores were scraped into sterile water to make a spore suspension as seed liquid; 2 ml of the seed liquid was added to 50 ml of fermentation medium, and cultured at 28°C, 150 r / min constant temperature shaking for 6 days. The obtained fermentation liquid was centrifuged at 6000 rpm / min and 4°C for 10 minutes, and the supernatant was placed in a 50 mL centrifuge tube, sealed and placed in a -20°C refrigerator for storage.

[0079] The cup-plate method was used to detect the antibacterial spectrum of the fermentation broth of strain JN4-2, comprising the following steps: preparing a 7 mm pathogen cake, symmetrically inverting the cake around the PDA culture medium, placing an Oxford cup in the center of the plate, inoculating the fermentation broth (i.e., the supernatant prepared by the above method) in the Oxford cup, the inoculation volume was 200 μL, and distilled water was used as a control, and the culture was cultured at a constant temperature of 28° C. After the bacteria in the control group filled the culture dish, the diameter of the antibacterial zone of the treatment group was measured by the cross method.

[0080] The tested pathogens were: 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 red star pathogen, blueberry branch blight pathogen, apple fruit rot pathogen, cucumber sclerotinia pathogen, and pear fruit rot pathogen.

[0081] The results showed that the antibacterial activity of the fermentation liquid of the antagonistic strain JN4-2 was determined by the cup-plate method, and the fermentation liquid still maintained a good antibacterial activity against the Korean pine blight pathogen ( Fig. 9 ) had the strongest antibacterial effect, reaching 34.60 mm, which was significantly different from the other tested strains; Fig.10 ) and apple rot bacteria, with the inhibition diameter reaching 31.92mm and 29.87mm respectively, and it has a strong antagonistic effect on eggplant brown streak pathogen and melon vine cut pathogen ( Fig.11 ), larch dieback pathogen ( Fig.12 ) also had a good inhibitory effect, with the inhibition diameters being 27.60mm, 27.07mm and 28.46mm respectively. The fermentation broth of the antagonistic bacteria had a significant inhibitory effect on all the tested pathogens (Table 3), among which the tobacco target spot pathogen had the smallest inhibition diameter, but still reached 18.25mm.

[0082] Table 3 Antibacterial spectrum of the fermentation broth of antagonistic bacteria JN4-2

[0083]

[0084] The data in the table are mean ± standard deviation.

[0085] In summary, the JN4-2 strain provided by the present invention has fungicidal activity, and its live bacteria and fermentation liquid both show strong inhibitory effects on Korean pine damping-off pathogens. Analysis may be due to the fact that the strain produces antifungal substances during its physiological metabolism.

[0086] The antagonistic strain JN4-2 obtained by the present invention has broad development prospects, a wide antibacterial spectrum, and great biological control potential. The active antibacterial components in its fermentation broth can be developed as biological pesticides for application in the prevention and treatment of various plant diseases after separation and purification. At the same time, compared with chemical pesticides, the fermentation broth of the 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.

[0087] 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 strain of Streptomyces fusiformis, characterized in that: The strain name is Streptomyces fusiformis ( Streptomyces netropsis )JN4-2, the deposit number is CGMCC No.28157.

2. A bacterial agent, characterized in that The invention comprises the Streptomyces fusiformis according to claim 1 or a fermentation product of the Streptomyces fusiformis according to claim 1.

3. Use of the Streptomyces fusiformis according to claim 1 and / or the bacterial agent according to claim 2 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.

4. Use of the Streptomyces fusiformis of claim 1 and / or the bacterial agent of claim 2 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.

5. The use of the Streptomyces fusiformis according to claim 1 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 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.

6. Use of the Streptomyces fusiformis described in claim 1 and / or the bacterial agent described in claim 2 in preventing and treating Korean pine damping-off disease.

7. A biological control method, characterized in that: The method comprises the following steps: using the fusiform streptomyces described in claim 1 and / or the microbial agent described in claim 2 for biological control, which is used for biological control of plant pathogenic bacteria, wherein the plant pathogenic bacteria are selected from one or more of the following: red pine damping-off pathogen, red bean anthracnose pathogen, aggregated burr hole fungus, apple shell monochromatic spore canker pathogen, corn leaf blight pathogen, tobacco target spot pathogen, rice seedling blight pathogen, melon vine cutting 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.

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