Streptomycete Y309 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses streptomycete Y309 and application thereof. The preservation number of the streptomyces sp. Y309 is GDMCC No: 64527, and the streptomyces sp. Y309 has relatively strong secondary metabolite synthesis capability and can be used for producing siderophore, IAA (Indoleacetic Acid) and the like. The strain Y309 also has the potential of plant growth promotion and disease control, and the strain Y309 has a relatively strong bacteriostatic effect on 12 pathogenic fungi such as fusarium oxysporum, rhizoctonia cerealis, monilinia fusarium and coracocystis fimbristipula as well as 7 pathogenic bacteria such as pseudomonas syringae lacrimation pathogenic variants and bacillus cereus, and has a relatively strong bacteriostatic effect on 12 pathogenic fungi such as fusarium oxysporum, rhizoctonia cerealis, monilinia fusarium and coracocystis fimbristipula. The strain Y309 has good prevention and treatment effects on wheat sharp eyespot, tomato gray mold and pepper fusarium wilt, fermentation liquor of the strain Y309 can remarkably promote wheat seed germination and seedling growth, agronomic characters such as the plant height, the dry weight and the leaf area in the corn seedling stage can be improved in different saline-alkali soil, and the strain Y309 has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a strain of Streptomyces Y309 and an application thereof. Background Art

[0002] Fungal plant diseases seriously impact global food security. Over 19,000 pathogenic fungi, primarily found in the Ascomycota and Basidiomycota phyla, are responsible for crop diseases worldwide. Fusarium oxysporum, Rhizoctonia graminearum, and Botrytis cinerea are among the world's most common pathogens, posing a threat to various regions of the world due to their wide host range, high incidence, and severe damage.

[0003] Fusarium wilt, caused by Fusarium oxysporum, is a serious vascular disease that infects a wide range of crops, including soybeans, bananas, cotton, tomatoes, and peppers. Long-term cropping, soil physical and chemical properties such as acidity and compaction, and greenhouse cultivation conditions such as high temperature and high humidity all contribute to the occurrence and aggravation of wilt. The incidence of pepper wilt is generally between 15% and 30%, occurring in all pepper-growing areas. Sheath blight, caused by Rhizoctonia graminearum, is a significant disease in wheat production. The area of ​​wheat sheath blight in my country is increasing annually, with incidence rates exceeding 70% in severely affected areas, resulting in yield reductions exceeding 30%. Gray mold, caused by Botrytis cinerea, is a common disease in tomato cultivation. It can infect the plant's stems, leaves, flowers, and fruits, resulting in annual yield losses of 20% to 50%.

[0004] Streptomyces are important resource microorganisms with complex secondary metabolic capabilities. Natural products derived from Streptomyces account for only 39% of currently isolated microbial secondary metabolites, but they produce nearly 80% of bioactive compounds. Streptomyces can directly or indirectly improve plant growth and development by producing plant hormones, improving soil nutrients, regulating plant metabolism, and enhancing plant stress resistance. Streptomyces from different sources exhibit diversity in natural product synthesis due to differences in their growth habitats and nutritional conditions. The development of Streptomyces resources with superior traits can effectively mitigate the harm to the ecological environment and human health caused by the indiscriminate use of chemical pesticides. Summary of the Invention

[0005] The present invention provides a strain of Streptomyces Y309 and its applications. Strain Y309 has a strong ability to synthesize secondary metabolites and exhibits broad-spectrum antibacterial activity, exhibiting strong inhibitory effects against common pathogenic fungi and bacteria. The present invention particularly relates to its application in the biological control of plant diseases such as pepper wilt, wheat sheath blight, and tomato gray mold.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a strain of Streptomyces having a biocontrol effect. Streptomyces sp. Y309, a new species of Streptomyces, was named Streptomyces sp., the strain was isolated from Aksu Prefecture, Xinjiang Uygur Autonomous Region in 2021 and has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, deposit number GDMCC No: 64527, deposit date April 19, 2024.

[0007] In a second aspect, the present invention provides a fermentation broth containing Streptomyces Y309, or a crude extract of the fermentation broth.

[0008] In a third aspect, the present invention provides a microbial agent, a biopesticide or a biofertilizer prepared from Streptomyces Y309, its fermentation broth or a crude extract of the fermentation broth.

[0009] In a fourth aspect, the present invention provides a biocontrol agent or antibacterial agent prepared by Streptomyces Y309.

[0010] In a fifth aspect, the present invention provides any of the following uses of Streptomyces Y309 or its bacterial agent: 1) Used to antagonize plant pathogenic fungi; 2) Used to antagonize plant pathogenic bacteria; 3) Used to prevent and control pepper wilt; 4) Used to prevent and control wheat sheath blight; 5) Used to prevent and control tomato gray mold; 6) Used for the preparation of biopesticides; 7) Used for preparing biofertilizer; 8) Used for plant growth promotion.

[0011] The plant pathogenic fungi of the present invention include but are not limited to Pseudomonas graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Fusarium moniliforme( Fusarium moniliforme ), Fusarium spp. ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia graminearum ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani )、Sweet potato long beak shell fungus( Ceratocystis fimbriata ), Alternaria solani ( Alternanria solani )、Brown rot fungus( Monilia laxa ), destroy anthrax ( Colletotrichum destructivum ), Septoria celeriac ( Septoria apiicola ).

[0012] The pathogenic bacteria include but are not limited to Pseudomonas syringae pv. Pseudomonas syringae pv. Lachrymans )、Xanthomonas campestris pv. capsici ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella sp.).

[0013] The plants mentioned in the aforementioned application 8) include but are not limited to wheat and pepper.

[0014] In a sixth aspect, the present invention provides any of the following uses of Streptomyces Y309 and / or the antibacterial active substances produced therefrom: a) for use against pathogens and plant diseases caused by pathogens; b) for inducing plants to enhance resistance to plant diseases caused by pathogens; c) Used for the preparation of biocontrol agents, antimicrobial agents or plant immunity inducers.

[0015] Furthermore, the pathogens include pathogenic fungi and pathogenic bacteria, such as the aforementioned plant pathogenic fungi and plant pathogenic bacteria.

[0016] Furthermore, the plant diseases include but are not limited to pepper wilt, wheat sheath blight, and tomato gray mold.

[0017] In a seventh aspect, the present invention provides any of the following applications of Streptomyces Y309: i) For the production of siderophores; ii) used to produce IAA; iii) used to dissolve organic phosphorus; iv) Used for hydrolysis of starch and cellulose; v) Used for coagulation and peptization of milk.

[0018] The present invention conducts multiphase identification on the antagonistic strain Streptomyces Y309 with broad-spectrum antibacterial activity isolated from the Aksu desert environment in Xinjiang. The strain has potential biocontrol application value and good development and application prospects.

[0019] The fermentation medium used for fermenting and culturing the Streptomyces Y309 described in the present invention is: 6.3 g / L yeast extract, 3.9 g / L glucose, 5.0 g / L xylose, 10.0 g / L soluble starch, 5 mg / L Cu(II), 1000 mL distilled water, and a pH of 7.2-7.4.

[0020] The present invention also relates to the stability and basic properties of the antifungal active substance of Streptomyces Y309. The antifungal substance exhibits stable activity at pH 4-9, after treatment at 60°C for 1 hour, and UV irradiation for 40 minutes. The n-butanol extract and fermentation broth of the strain exhibited no significant difference in inhibition against Fusarium oxysporum, with rates of 61.2% and 63.4%, respectively. The crude protein extract and ethyl acetate extract showed weak activity.

[0021] Strain Y309 had a strong inhibitory effect on the mycelial growth of the 12 plant pathogenic fungi tested, among which the inhibitory effect on Rhizoctonia graminearum, brown rot fungus, and sweet potato long beak Aspergillus was the strongest, with inhibition rates of 80.9%, 76.1%, and 71.1%, respectively. The inhibition rates on the other pathogenic fungi, Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Fusarium spp., Botrytis cinerea, Rhizoctonia solani, Alternaria solani, and Colletotrichum destructans were all over 60%, and the inhibition rate on Septoria acerifolia was the lowest, only 50.2%. Strain Y309 exhibited the strongest inhibitory activity against Pseudomonas syringae pv. lacrimalis and Bacillus cereus, with inhibition zone diameters exceeding 30 mm, at 34.75 mm and 32.17 mm, respectively. The inhibition zone diameters ranged from 5.23 to 22.80 mm against Xanthomonas campestris pv. capsici, R. solanacearum, Xanthomonas citri subsp. citri, Staphylococcus aureus, and Salmonella. However, it had no inhibitory effect against Escherichia coli, Agrobacterium tumefaciens, and Pectobacterium carotovora. Greenhouse pot experiments demonstrated that strain Y309 had control efficacy of 52.8%, 65.1%, and 40.0% against the stem base disease wheat sheath blight, the leaf disease tomato gray mold, and the root disease pepper wilt, respectively. The fermentation broth of strain Y309 significantly promoted wheat growth, increasing seed germination and seedling dry weight by 11.7% and 23.9%, respectively. Strain Y309 has great potential in plant disease prevention and control and has good application prospects in biological pesticides and antibacterial agents.

[0022] The Streptomyces Y309 of the present invention has a strong ability to synthesize secondary metabolites and can produce siderophores, IAA, etc. When the fermentation medium formula is 6.3 g / L yeast extract, 3.9 g / L glucose, 5.0 g / L xylose, 10.0 g / L soluble starch, 5 mg / L Cu(II), and 1000 mL distilled water, the fermentation broth has the strongest activity against Fusarium oxysporum. The activity of the active substance is stable at pH 4-9 after being treated at 60°C for 1 hour and irradiated with ultraviolet light for 40 minutes, indicating that the bacterium has the potential for plant growth promotion and disease prevention. Strain Y309 has a strong antibacterial effect against 12 pathogenic fungi such as Fusarium oxysporum, Rhizoctonia graminearum, brown rot fungus, and sweet potato long-beaked Aspergillus, as well as 7 pathogenic bacteria such as Pseudomonas syringae pv. salina and Bacillus cereus. It has a good preventive and control effect on wheat sheath blight, tomato gray mold and pepper wilt. The fermentation liquid of strain Y309 can significantly promote wheat seed germination and seedling growth, and can improve agronomic traits such as plant height, dry weight, and leaf area of ​​corn seedlings in different saline-alkali soils, indicating that the bacterium has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a phylogenetic tree constructed based on the 16S rRNA gene of strain Y309 in a preferred embodiment of the present invention and similar model strains of Streptomyces.

[0024] Figure 2 This is a phylogenetic tree based on the housekeeping gene phylogeny of strain Y309 in a preferred embodiment of the present invention and similar model strains of Streptomyces.

[0025] Figure 3 This is a phylogenetic tree based on the whole genome sequence of strain Y309 in a preferred embodiment of the present invention and similar model strains of Streptomyces.

[0026] Figure 4 Figures 1 and 2 show the morphological characteristics of strain Y309 in a preferred embodiment of the present invention. (a) Culture characteristics of strain Y309 (left: back of colony; right: front of colony); (b) Optical micrograph of strain Y309; (c) Scanning electron micrograph of strain Y309.

[0027] Figure 5 These are the physiological and biochemical characteristics of strain Y309 in a preferred embodiment of the present invention.

[0028] Figure 6 Figure 2 shows the effects of different culture media on the growth and antibacterial activity of strain Y309 in a preferred embodiment of the present invention. (a) Antibacterial rate of different fermentation media; (b) Colony counts of Fusarium oxysporum in different fermentation media at 1% addition; (c) Antibacterial rate of different fermentation media at 1% addition; (d) Biomass per treatment group in different fermentation media at 1% addition.

[0029] Figure 7 The carbon source optimization of strain Y309 in the preferred embodiment of the present invention is shown in Figure 1. ① to ⑨ are lactose, galactose, soluble starch, sucrose, glycerol, fructose, maltose, ISP2, and Natamycin.

[0030] Figure 8 The nitrogen source optimization of strain Y309 in the preferred embodiment of the present invention is as follows: ① to ⑨ are soybean meal extract, peptone, soy peptone, tryptone, beef extract powder, ammonium sulfate, ammonium nitrate, ISP2, and Natamycin.

[0031] Figure 9 This is the trace element optimization of strain Y309 in the preferred embodiment of the present invention.

[0032] Figure 10 The influencing factors of strain Y309 in the preferred embodiment of the present invention are optimized. ① to ⑥ are xylose, methionine, cysteine, tyrosine, ISP2, and Natamycin, respectively.

[0033] Figure 11 ① to ③ are respectively the unoptimized ISP2 medium (ISP), the optimal medium predicted by the CCD model (UISP), and Natamycin.

[0034] Figure 12 This is the antibacterial concentration gradient of the fermentation broth of strain Y309 in a preferred embodiment of the present invention.

[0035] Figure 13 The antibacterial ability of the fermentation broth of strain Y309 under different acid and alkali treatment conditions in the preferred embodiment of the present invention is shown.

[0036] Figure 14 The antibacterial ability of the fermentation broth of strain Y309 under different temperature treatment conditions in the preferred embodiment of the present invention.

[0037] Figure 15 The antibacterial ability of the fermentation broth of strain Y309 under different UV irradiation times in the preferred embodiment of the present invention.

[0038] Figure 16 This is the antibacterial ability of the crude protein extract of strain Y309 in the preferred embodiment of the present invention.

[0039] Figure 17 This is the antibacterial ability of the crude lipopeptide extract of strain Y309 in a preferred embodiment of the present invention.

[0040] Figure 18Figures a through e show the antibacterial activity of the organic extract and aqueous phase of strain Y309 in a preferred embodiment of the present invention. Figures a through e show the diameters of Fusarium oxysporum colonies generated when the extract / aqueous phase was concentrated 20-fold and added to a PDA plate. "No. 1" represents the first organic solvent extraction; "No. 2" represents the second organic solvent extraction; "No. 3" represents the third organic solvent extraction; and "No. 4" represents the fourth organic solvent extraction.

[0041] Figure 19 This is the antagonism of strain Y309 against pathogenic fungi in a preferred embodiment of the present invention.

[0042] Figure 20 This is the antagonism of strain Y309 against pathogenic bacteria in a preferred embodiment of the present invention.

[0043] Figure 21 This figure shows the growth-promoting effect of strain Y309 on wheat in a preferred embodiment of the present invention. "1×" indicates a stock solution of sterile fermentation broth; "100×" indicates a 100-fold dilution of sterile fermentation broth; "500×" indicates a 500-fold dilution of sterile fermentation broth; and "1000×" indicates a 1000-fold dilution of sterile fermentation broth.

[0044] Figure 22 The control ability of strain Y309 against wheat sheath blight in a preferred embodiment of the present invention is shown in Table 1. “1×” indicates the original sterile fermentation solution; “5×” indicates the sterile fermentation solution diluted 5 times.

[0045] Figure 23 The control ability of strain Y309 against tomato gray mold in a preferred embodiment of the present invention is shown in Table 1. “1×” indicates the original sterile fermentation broth; “5×” indicates the sterile fermentation broth diluted 5 times.

[0046] Figure 24 This is the ability of strain Y309 in a preferred embodiment of the present invention to prevent and control pepper wilt.

[0047] Figure 25 This is the growth-promoting effect of strain 309 on corn in saline-alkali soil in Xinjiang in a preferred embodiment of the present invention.

[0048] Figure 26 This is the growth-promoting effect of strain Y309 on corn in Xinjiang non-saline-alkali soil with added salt in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0049] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0050] The test bacteria involved in the following examples are as follows, all of which are from China Agricultural University: Pathogenic fungus: Pseudomonas graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Fusarium moniliforme( Fusarium moniliforme ), Fusarium spp. ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia graminearum ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani )、Sweet potato long beak shell fungus( Ceratocystis fimbriata ), Alternaria solani ( Alternanria solani )、Brown rot fungus( Monilia laxa ), destroy anthrax ( Colletotrichum destructivum ), Septoria celeriac ( Septoria apiicola ).

[0051] Pathogen: Pseudomonas syringae p ... Pseudomonas syringae pv. Lachrymans )、Xanthomonas campestris pv. capsici ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Agrobacterium tumefaciens ( Agrobacterium vitis )、Carrot Pectin Bacillus( Pectobacterium carotovorum ), Bacillus cereus ( Bacillus cereus ) Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ),salmonella( Salmonella sp.).

[0052] Example 1 Identification of strain Y309 1. Isolation and purification of strain Y309 In 2021, soil samples were collected in Aksu, Xinjiang Uygur Autonomous Region. 10 g of soil sample was weighed and added to a conical flask containing 100 mL of sterile saline. After standing for 20 min, it was shaken on a shaker at 28°C and 150 rpm for 30 min and then stood for 10 min. 100 μL of supernatant and 900 μL of sterile saline were pipetted and added to a 1 mL centrifuge tube for dilution. The sample was then diluted 10 min later. 2 , 10 3 , 10 4100 μL of each soil suspension was spread on a plate containing 0.01% potassium dichromate containing Gao's medium No. 1 (20 g soluble starch, 1 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.5 g magnesium sulfate heptahydrate, 0.01 g ferrous sulfate heptahydrate, 1000 mL distilled water, pH 7.2–7.4). The plate was incubated at 28°C for 5 days. A single bacterial colony was picked with a sterile toothpick, streaked, purified, and stored for later use.

[0053] 2. Identification of strain Y309 Strain Y309 was identified and described using a multi-phase identification method, including molecular biological identification and phenotypic characteristics observation, and was shown to be a potential new species of Streptomyces. The details are as follows: (1) 16S rRNA and housekeeping gene sequence analysis: The genomic DNA of actinomycetes was extracted using a bacterial genome rapid extraction kit. The genomic DNA of Y309 strain was used as a template and universal primers (Table 1) were used to amplify the 16S rRNA (SEQ ID NO: 1) and atpD 、 gyrB 、 recA 、 rpoB 、 trpB PCR amplification of five housekeeping genes, including SEQ ID NO: 2-6, was performed. The amplification reaction system and conditions are shown in Tables 2 and 3. The sequence after sequencing and splicing was compared with all known gene sequences in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) for similarity online analysis. Subsequently, the Neighbor-Joining Method (NJ) in MEGA 11 software was used to construct the 16S rRNA and housekeeping gene sequences. atpD - gyrB - recA - rpoB - trpB The concatenated phylogenetic trees were used to determine the phylogenetic relationship between strain Y309 and related strains.

[0054] Table 1 PCR primers used in this experiment

[0055] Table 2 PCR reaction system

[0056] Table 3 PCR reaction conditions The results are as follows Figure 1 、 Figure 2 As shown, the Y309 strain and S. aureoverticillatusJCM4347 T The phylogenetic relationship is the closest, with a homology of up to 99.89%, but the sequence similarity of the five housekeeping genes is between 94.30% and 97.86%, among which gyrB The gene sequence similarity was 96.57%, which was lower than the recommended threshold of 98.5% for new species classification. It was speculated that the Y309 strain might be a potential new species of Streptomyces.

[0057] (2) Whole genome sequence analysis The Y309 strain was sent to Beijing Aoweisen Gene Technology Co., Ltd. for whole-genome sequencing using a combination of second-generation Illumina and third-generation PacBio sequencing. The Y309 strain was compared with all known strains in the database using the online platforms JSpecies Web Server (http: / / jspecies.ribohost.com / jspeciesws / ) and Type(Strain)Genome Server (TYGS, https: / / tygs.dsmz.de / ). The average nucleotide identity (ANI) and DNA hybridization (DDH) values ​​between the two strains were calculated, and a phylogenetic tree of the whole-genome sequence was constructed.

[0058] The results are as follows Figure 3 As shown in the figure, the total genome size of strain Y309 is 10.05 Mb, the genome (G+C) mol% content is 71.75%, and it consists of one chromosome and three plasmids. The linear chromosome size is 9.35 Mb, and 8410 coding genes are predicted, accounting for 85.7% of the total genome. The phylogenetic tree separates this bacterium from S. aureoverticillatus JCM4347 T Divided into one ( Figure 3 a in the figure), but the dDDH and ANIb values ​​between the Y309 strain and similar standard strains were between 22.6~45.1% and 77.4~91.8% ( Figure 3 b), while the Y309 strain is the closest related standard strain S. aureoverticillatus JCM4347 T The dDDH and ANIb values ​​between the two groups were 45.1% and 91.8%, respectively, which were both lower than the thresholds for new species classification (70% and 95%), proving that Streptomyces sp. Y309 is a new species in the genus Streptomyces.

[0059] (3) Phenotypic characteristic analysis The strain was inoculated onto seven ISP media from the International Streptomyces Project, as well as NA and Czapek's media, and cultured at 28°C for 21 days to observe characteristics such as matrix hyphae, aerial hyphae, and soluble pigment. The strain was inoculated onto ISP2 medium and cultured at 28°C for 14 days. Slides were prepared and examined for hyphal morphology, spore chain morphology, and spore surface morphology under a light microscope (BX53F, Olympus, Japan) and an electron microscope (SU8100, Hitachi, Japan).

[0060] The growth of strain Y309 was tested on ISP2 solid medium, a well-established growth medium, at different temperatures (4, 10, 20, 28, 37, 45, 50, and 55°C) for 7 days. The strain was inoculated onto ISP2 medium containing varying pH values ​​(4.0–12.0) and salt concentrations (0–10%, w / v%) and cultured at 28°C for 7 days to assess its pH and salt tolerance.

[0061] To test the IAA production capacity of the strain, Streptomyces Y309 was streaked onto PDA plates for activation. After 7 days of incubation at 28°C, a single colony was punched out using a 5 mm diameter borer and inoculated into liquid PD medium. The culture was shaken at 28°C and 150 rpm for 7 days. After 7 days, the culture suspension was removed and centrifuged at 12,000 rpm for 5 minutes. The IAA content in the suspension was determined using the Salkowkin colorimetric method in triplicate. The results showed that Y309 produced 7.33 ± 0.02 mg / L of IAA.

[0062] Other physiological and biochemical characteristics, such as starch hydrolysis, gelatin liquefaction, and nitrate reduction, were performed according to the protocols in the "Common Bacterial Systematic Identification Manual." Using the Biolog Gen III bacterial identification kit, the plates were incubated at 28°C for 48 hours. During incubation, the microplates were read multiple times using an automated microbial rapid identification instrument (GEN III, USA). The Y309 strain was tested for its utilization of 71 carbon sources, including dextrin and maltose, and its sensitivity to 23 chemicals, including sodium lactate and lincomycin. The activity of 19 enzymes, including alkaline phosphatase and esterase, was measured using API ZYM kits.

[0063] The morphological characteristics of Y309 strain on different culture media are shown in Figure 4The results showed that strain Y309 grew well on ISP2, ISP3, ISP4, ISP7, and Czapek's medium, but grew poorly on ISP1, ISP5, ISP6, and NA medium. No soluble pigment production was observed on any of the nine media. Furthermore, strain Y309 exhibited different colony morphology, matrix mycelium and aerial mycelium color, and conidia production on different media. Specific characteristics are shown in Table 4. Microscopic observation of strain Y309 revealed typical Streptomyces characteristics, including well-developed branched hyphae, long, chain-like spore chains, cylindrical spores measuring 0.8 to 1.1 µm, smooth surfaces, and the absence of specialized structures such as spiny or scaly structures ( Figure 4 , b and c).

[0064] Table 4 Morphological characteristics of strain Y309 grown on different culture media

[0065] The Y309 strain can grow in temperatures ranging from 15°C to 45°C, a pH range of 6 to 11, and a NaCl tolerance range of 0% to 8%. The Y309 strain can produce siderophores, IAA, and solubilize organic phosphorus, but cannot produce H2S. It can hydrolyze starch and cellulose, coagulate and peptize milk, but cannot hydrolyze esculin. Furthermore, the strain tested positive for urease, lipase, protease, oxidase, and catalase, but negative for citrate utilization, nitrate reduction, gelatin liquefaction, MR, and VP. Figure 5 ).

[0066] The results of API ZYM enzyme activity test showed that the bacteria had enzyme activities such as alkaline phosphatase, leucine arylaminease, valine arylaminease, cystine arylaminease, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-uronidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase, but did not have enzyme activities such as esterase (C4), lipid esterase (C8), lipase (C14) and chymosin.

[0067] Biolog Gen Ⅲ test results showed that the Y309 strain can utilize trehalose, gentianose, turanose, lactose, glucose, mannose, melibiose, fructose, galactose, rhamnose, and glycerol as sole carbon sources, but cannot utilize maltose, cellobiose, raffinose, salicin, sucrose, mannitol, sorbitol, inositol, and adenosine. It can utilize nitrogen sources such as L-alanine, L-arginine, L-glutamic acid, and L-serine, but cannot utilize nitrogen sources such as L-aspartic acid, D-serine, and D-aspartic acid. In addition, the strain can also utilize various hexonic acids such as D-galacturonic acid and D-glucuronic acid, various carboxylic acids such as malic acid and acetic acid, fatty acids such as acetoacetic acid, and lipids such as D-methyl lactate. The strain is sensitive to substances such as vancomycin, sodium butyrate, and minocycline, but can grow in the presence of substances such as sodium lactate, rifamycin, nalidixic acid, and aztreonam.

[0068] The strain Y309 in the present invention and the closely related model strains S. aureoverticillatus JCM4347 T There were significant differences in colony color between the strains. The reduction of nitrate, coagulation and peptidation of milk, and utilization of carbon sources such as sucrose, L-arabinose, and raffinose became the main basis for distinguishing the Y309 strain from its closely related type strains (Table 5).

[0069] Table 5 Differential characteristics between strain Y309 and similar strains of Streptomyces Example 2 Fermentation conditions of strain Y309 The present invention provides a fermentation culture method suitable for high-yield antibacterial substances of the Y309 strain. The optimized conditions include carbon source, nitrogen source, trace elements, and influencing factors. The antibacterial activity of the fermentation broth against Fusarium oxysporum is used as the measurement indicator, and the optimal culture medium is predicted through a central combination experimental design.

[0070] (1) Preparation of seed solution Streptomyces Y309 strain was selected and streaked on a PDA plate for activation. After culturing in a constant temperature incubator at 28°C for 7 days, a single colony cake was punched out with a 5 mm diameter punch and inoculated into 150 mL of liquid PD medium. The culture was then incubated at 28°C and 150 rpm for 5 days to prepare the seed solution.

[0071] (2) Fermentation conditions 5 mL of the supernatant without obvious mycelium in the seed solution was inoculated into 150 mL of culture medium and cultured at 28 °C and 150 rpm for 7 days to obtain the fermentation liquid. Each treatment was repeated 3 times.

[0072] (3) Growth assay of strain Y309 The dry weight of mycelium was used to characterize the growth of the strain. The fermentation broth was centrifuged at 10,000 rpm for 10 min. The bacterial pellet was rinsed 2 to 3 times with sterile water and then dried in an oven at 60°C to constant weight. The dry weight of the mycelium under different culture conditions was recorded, and the supernatant after centrifugation was used for subsequent activity detection.

[0073] (4) Activity determination of strain Y309 fermentation broth After centrifugation, the fermentation supernatant was sterilized by filtration through 0.45 µm and 0.22 µm sterile filter membranes, respectively. The sterile fermentation broth was stored in a refrigerator at 4°C in the dark. The growth inhibition of the fermentation broth against Fusarium oxysporum was determined using the drug-containing plate assay and the inhibition zone assay.

[0074] Antibacterial activity was determined using the drug-containing plate method. Sterile fermentation broth was added to pre-thawed PDA medium cooled to approximately 50°C. Gently shaken until thoroughly mixed, the mixture was then poured into a 60 mm diameter Petri dish to create a drug-containing plate with a final fermentation broth concentration of 1% to 5%. A 5 mm thick cake of pathogenic fungi was seeded in the center of the plate and incubated at 28°C for 3 days. Colony diameters were measured using the cross-hatch method, and the inhibition rate was calculated according to Formula 1). The inhibition rate reflects the strength of the antibacterial activity and the amount of the inhibitory substance present.

[0075] The antibacterial activity was determined by the inhibition zone method. 200 μL of 1.0×10 6 A spore suspension of 100 cfu / mL was evenly spread using sterile glass beads. A 7 mm sterile hole punch was used to evenly punch wells in the plate. The agar blocks were removed and 100 µL of sterile fermentation broth was added to the wells. The culture medium was refrigerated at 4°C overnight. After the liquid in the wells was completely absorbed, the culture was transferred to a 28°C incubator and incubated for 36 hours. The diameter of the clear zone was measured using the cross-hatch method and the diameter of the inhibition zone was calculated according to Formula 2, using 30 µg / mL of natamycin as a correction.

[0076] (5) Culture medium optimization The drug-containing plate method was used to determine the growth inhibitory effects of six different fermentation media, including ISP2, YM, YMG, T6, Gao's medium No. 1, and Czapek's medium, on Fusarium oxysporum to screen the most suitable medium for the strain to produce antibacterial substances. The medium formula is as follows: PDB medium: 200.0 g potato, 20.0 g glucose, 1000 mL distilled water, natural pH.

[0077] ISP2 medium (Yeast extract-malt extract agar): yeast extract 4.0 g, glucose 4.0 g, malt extract 10.0 g, distilled water 1000 mL, pH 7.2-7.4.

[0078] Czapek Dox Medium (hereinafter referred to as Czapek): 2.0 g sodium nitrate, 0.5 g potassium chloride, 30.0 g sucrose, 1.0 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1000 mL distilled water, pH 7.0-7.4.

[0079] Gauze's Synthetic Medium No. 1 (Gauze's): 20.0 g soluble starch, 1.0 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.5 g magnesium sulfate heptahydrate, 0.01 g ferrous sulfate heptahydrate, 1000 mL distilled water, pH 7.2-7.4.

[0080] YMG medium: 4.0 g glucose, 4.0 g yeast extract, 10.0 g malt extract, 1000 mL distilled water, pH 7.2.

[0081] YM medium: yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, glucose 10.0 g, distilled water 1000 mL, pH 5.0-6.0.

[0082] T6 medium: 10.0 g soluble starch, 10.0 g glucose, 10.0 g glycerol, 5.0 g tryptone, 5.0 g yeast extract, 1000 mL distilled water, pH 7.0.

[0083] The results are as follows Figure 6 As shown, ISP2 medium was used as the initial fermentation medium for Y309 strain for subsequent optimization.

[0084] (6) Carbon source optimization The carbon sources were replaced by lactose, galactose and soluble starch in equal amounts instead of malt extract powder; glucose was replaced by sucrose, glycerol, fructose and maltose in equal amounts, and the other ingredients were kept consistent with the control. The antibacterial effect of the fermentation broth on Fusarium oxysporum was determined.

[0085] The results are as follows Figure 7 As shown in the results, different carbon sources had significant effects on the activity of the strains. When lactose and soluble starch were used to replace the yeast extract in the initial culture medium, the antibacterial activity of the fermentation broth was significantly increased. Considering the antibacterial activity and economic value of the strain, soluble starch was selected for subsequent optimization studies.

[0086] (7) Nitrogen source optimization On the basis of the optimal carbon source, the nitrogen source was replaced by equal amounts of soybean meal extract, peptone, soy peptone, tryptone, beef extract powder, (NH4)2SO4, and NaNO3 respectively, and the other ingredients were kept consistent with the control. The antibacterial effect of the fermentation broth on Fusarium oxysporum was determined.

[0087] The results are as follows Figure 8 As shown in the results, the antibacterial activity was the highest when the initial yeast extract was used as the nitrogen source, which was 22.79±0.78 mm. Therefore, the initial nitrogen source yeast extract was still selected for subsequent optimization studies.

[0088] (8) Trace element optimization Ten trace element salt ions (chloride salts) were exogenously added to the culture medium: Fe(Ⅱ), Mn(Ⅱ), Cu(Ⅱ), Zn(Ⅱ), Co(Ⅱ), Ni(Ⅱ), V(Ⅲ), Se(Ⅳ), Mo(Ⅴ), and W(Ⅵ). The concentration gradients were set at 1, 5, 10, 15, and 20 mg / L, respectively. The control group without the addition of the above trace elements was used to determine the antibacterial effect of the fermentation broth on Fusarium oxysporum.

[0089] The results are as follows Figure 9 As shown in the results, the addition of Cu(Ⅱ) significantly improved the antibacterial activity of the strain, and there was no significant difference between the addition amounts of 5, 10, and 15 mg / L Cu(Ⅱ), so 5 mg / L Cu(Ⅱ) was selected for subsequent optimization experiments.

[0090] (9) Impact factor optimization Based on the optimal carbon source, nitrogen source and trace elements, 100 mg / L methionine, cysteine, tyrosine and 5 g / L xylose (a pentose that may affect the metabolic process) were screened, and the antibacterial effect of the fermentation broth against Fusarium oxysporum was determined.

[0091] The results are as follows Figure 10 As shown, xylose was incorporated into the fermentation medium of the strain for subsequent optimization.

[0092] (10) Plackett-Burman experimental design To determine the influence of the selected culture medium components, the diameter of the fermentation broth inhibition zone was used as the response value and the PB experiment was designed using Design-expert 13.0. The 5-factor 2-level design is shown in Table 6.

[0093] Table 6 Plackett-Burman experimental design factors and levels

[0094] The results of the PB experiment are shown in Table 7. The complete loss of antibacterial activity of multiple fermentation broth components is all due to the high "1" level of xylose, indicating that the xylose concentration of 8 g / L is not suitable for the growth and fermentation of the strain. Therefore, the effect of xylose concentration on the other two significant factors cannot be ignored. The regression analysis of the response value results is shown in Table 8, and the regression equation for the influence of each factor on the response value can be obtained: Y=48.95-2.73A-0.26B+2.94C-0.19D-5.83E+0.35AE-0.35CE. And the variance analysis shows that P Xylose (E) < 0.0001 is the most important factor affecting the activity of the strain, followed by glucose ( P =0.0254) and yeast extract ( P =0.0281). The prediction curve showed that yeast extract and xylose had a significant negative effect on the response value, while the addition of glucose had a positive effect on the response value.

[0095] Table 7 Plackett-Burman experimental design and results

[0096] Table 8 Plackett-Burman analysis of variance (11) Steepest climbing test According to the Plackett-Burman experiment, the factors that significantly affect the antibacterial activity of strain Y309 were screened out. In order to determine the positive and negative effects of the significant factors, the appropriate step size and climbing direction were set so that the addition amount of the significant factors approached the optimal area, the center point was found for the response surface model, and the optimal addition amount of each significant factor was determined.

[0097] The results are shown in Table 9. The corresponding factor levels of Experiment 3 can maximize the activity of strain Y309, so this addition amount is selected as the center point of the subsequent central combination experiment.

[0098] Table 9 Steepest climbing experiment design and results

[0099] (12) Central combination design of culture medium components Design—Expert 13.0 was used to design the Central Composite experiment. Each factor took five levels: -2, -1, 0, 1, and 2. A multiple regression equation was established and the optimal concentration value of each factor was predicted, providing a feasible solution for strains with high antibacterial activity in liquid fermentation (Table 10).

[0100] Table 10 Central Composite Experimental Design Factors and Levels

[0101] A central composite design (CCD) experiment using response surface methodology (RSM) was conducted using 20 experimental combinations at five different levels to determine the optimal values ​​of the variables that significantly affected the antibacterial activity of the strain. The experimental design and results are shown in Table 11. Regression analysis of the response value results (Table 12) yielded a nonlinear regression equation for the effect of each factor on the diameter of the inhibition zone: Y = 26.49 - 2.48A - 1.82B - 2.79C - 3.33AB - 0.6475AC + 1.0BC - 1.81A 2 -0.3384B 2 -0.9719C 2 The model parameters demonstrated its effectiveness and indicated that the model could be used to predict the antibacterial activity of Y309 fermentation broth. Response surface optimization experiments predicted the most suitable culture medium for Y309 fermentation as follows: yeast extract 6.3 g / L, glucose 3.9 g / L, xylose 5.0 g / L, soluble starch 10.0 g / L, and Cu(Ⅱ) 5 mg / L.

[0102] Table 11 Central composite experimental design and results

[0103] Table 12 Analysis of variance for central composite experimental design 13) Fermentation optimization verification experiment The optimal fermentation medium combination obtained from the response surface model was verified by shaking culture fermentation, and three replicates were set to verify the accuracy and feasibility of the response surface model.

[0104] The results are as follows Figure 11 As shown in the results, the optimized culture medium significantly improved the antibacterial activity of the fermentation broth of strain Y309 against Fusarium oxysporum, which was increased by 103.3% compared with the unoptimized level. The diameter of the inhibition zone reached 31.10 mm, which was a very significant difference. In addition, the strain grew more efficiently, with a biomass of 0.85 g, which was 21.2% higher than before optimization.

[0105] Example 3 Extraction and stability analysis of crude extracts of strain Y309 (1) Preparation of fermentation broth The fermentation medium was the optimal medium predicted by the response surface method in Example 2, with the following formulation: yeast extract 6.3 g / L, glucose 3.9 g / L, xylose 5.0 g / L, soluble starch 10.0 g / L, Cu(II) 5 mg / L, distilled water 1000 mL, pH 7.2-7.4. The fermentation method was the same as in Example 2.

[0106] (2) IC50 of the fermentation broth against Fusarium oxysporum 50 Determination The drug-containing plate method was used to determine the inhibition rate of different fermentation broth concentrations from 0% to 5%. The logistic equation was fitted using Origin 2021 to obtain the dose-effect curve of the aseptic fermentation broth against Fusarium oxysporum, and the IC was calculated. 50 The results are as follows Figure 12 As shown, the half inhibitory concentration IC of the fermentation broth of strain Y309 against Fusarium oxysporum 50 is 5.05 µL / mL.

[0107] (3) Acid-base stability of antibacterial substances of strain Y309 The pH of the fermentation broth was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, and 10 with 1 mol / L HCl and NaOH solutions, respectively. After standing at room temperature for 24 h, it was adjusted to the original state. The untreated sterile fermentation broth was used as the control. The effects of different acid and alkaline conditions on the antibacterial substances of strain Y309 were determined by the drug-containing plate method. The amount of drug solution added to the drug-containing plate was the calculated IC 50 The concentration is 5.05 µL / mL. Figure 13 As shown, the antibacterial substances in the fermentation broth have high acid-base stability. When the pH value is 4-9, their antibacterial activity is not significantly different from that of the original sterile fermentation broth. This shows that the antibacterial substances synthesized by Y309 have strong acid-base stability.

[0108] (4) Temperature stability of antibacterial substances of strain Y309 The fermentation broth was heated in a water bath at 40, 60, 80, and 100 °C for 1 h, and then treated in a high-pressure steam sterilizer at 121 °C for 20 min. After the fermentation broth returned to room temperature, the untreated sterile fermentation broth was used as a control. The effects of different temperature treatments on the antibacterial substances of strain Y309 were determined using the drug-containing plate method. The amount of drug solution added to the drug-containing plate was the calculated IC 50 The concentration is 5.05 µL / mL. Figure 14 As shown in the results, the antibacterial activity of the fermentation broth did not change significantly after being treated at 40-60°C for 1 h, while the activity of the fermentation broth remained around 80% after being treated at 80°C for 1 h. This indicates that the antibacterial substance synthesized by Y309 has strong temperature tolerance and stability.

[0109] (5) UV stability of antibacterial substances of strain Y309 The sterile fermentation liquid was placed under an ultraviolet lamp (30 W) at a distance of 20 cm and irradiated for 20, 40, 60, 90, and 120 min, respectively. The untreated sterile fermentation liquid was used as a control. The effect of different ultraviolet irradiation times on the antibacterial substances of strain Y309 was determined using the drug-containing plate method. The amount of drug solution added to the drug-containing plate was the calculated IC 50 The concentration is 5.05 µL / mL. Figure 15 As shown in the figure, the activity of the fermentation broth decreased significantly after being exposed to UV for more than 40 min. As the UV irradiation time increased, the antibacterial activity of the fermentation broth continued to decrease. After 120 min of irradiation, the activity of the fermentation broth was only about 40% of the original fermentation broth.

[0110] (6) Activity determination of crude water-soluble protein extracts of strain Y309 The water-soluble protein substances in the fermentation broth of strain Y309 were extracted using ammonium sulfate precipitation. The precipitate was dissolved with 0.01 M PBS, dialyzed in a 3500 D dialysis bag for desalination, and sterilized by filtration through a 0.22 μm sterile filter membrane to obtain a crude water-soluble protein extract. The protein content was determined using Coomassie Brilliant Blue G-250. After gradient dilution, the protein content was 1, 5, 10, 15, and 20 times that of the original fermentation broth. The antibacterial activity of the solution was determined using the drug-containing plate method. The amount of drug solution added to the drug-containing plate was the calculated IC 50 The crude extract was tested for antibacterial activity at a concentration of 5.05 µL / mL. Figure 16 As shown in the results, although the protein substances in the fermentation broth of strain Y309 have certain activity against Fusarium oxysporum, indicating that Y309 synthesizes protein-based antibacterial substances, it is not the only antibacterial substance.

[0111] (7) Activity determination of crude water-soluble lipopeptide extracts from strain Y309 Lipopeptides were extracted from the fermentation broth of strain Y309 using the acid precipitation and alcohol extraction method. The precipitate was extracted with 10% methanol by ultrasonic assisted extraction and filtered through a 0.22 µm sterile filter membrane to obtain a water-soluble lipopeptide crude extract. After gradient dilution, solutions with volumes concentrated to 1, 5, 10, 15, and 20 times that of the original fermentation broth were obtained. The antibacterial activity of the solution was determined using the drug-containing plate method. The amount of drug solution added to the drug-containing plate was the calculated IC 50 The crude extract was tested for antibacterial activity at a concentration of 5.05 μL / mL. Figure 17 As shown in the figure, crude lipopeptide extracts at different dilution ratios showed no antibacterial activity against Fusarium oxysporum, indicating that the antibacterial substance synthesized by Y309 is a non-lipopeptide substance.

[0112] (8) Activity determination of small molecule crude extracts of strain Y309 The different polar small molecules in the fermentation broth of strain Y309 were extracted four times in a row using two volumes of petroleum ether, toluene, ethyl acetate, and water-saturated n-butanol. The organic phases of the four extraction processes were collected and the final aqueous phase was saved. The organic phase was evaporated to dryness on a rotary evaporator at 60°C and gradiently diluted with 10% methanol to obtain solutions with volumes concentrated to 1, 5, 10, 15, and 20 times that of the original fermentation broth. The antibacterial activity of each phase was determined using the drug-containing plate method. The amount of drug solution added to the drug-containing plate was IC 50 The crude extract was tested for antibacterial activity at a concentration of 5.05 µL / mL. Figure 18 As shown, the active substances in the fermentation broth of strain Y309 are small molecules with medium or even high polarity, with activity concentrated in the n-butanol extract. Further investigation of the specific active substances in the crude n-butanol extract is required. This suggests that the antimicrobial substances synthesized by Y309 are readily soluble in n-butanol, but have low solubility in petroleum ether, toluene, and ethyl acetate.

[0113] Example 4 Broad-spectrum antibacterial effect of strain Y309 (1) Antibacterial ability of strain Y309 against pathogenic fungi The plate stand-off method was used to determine the Y309 strain's resistance to Fusarium graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Fusarium moniliforme( Fusarium moniliforme ), Fusarium spp. ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia graminearum ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani )、Sweet potato long beak shell fungus( Ceratocystis fimbriata ), Alternaria solani ( Alternanria solani )、Brown rot fungus( Monilia laxa ), destroy anthrax ( Colletotrichum destructivum ), Septoria celeriac ( Septoria apiicola ) were tested for growth inhibition against 12 pathogenic fungi from eight genera, including 12 strains. Y309 strains were picked with a sterile toothpick and inoculated 25 mm apart on either side of a PDA plate. After incubation at 28°C for 5 days, a 5 mm thick cake of pathogenic fungus was inoculated at the center of two Y309 colonies. A plate inoculated with the pathogen alone served as a control. The plate was incubated in a 22°C incubator until the control nearly filled the plate. The diameter of the pathogenic fungus was then measured, and the growth inhibition rate was calculated according to Formula 1.

[0114] The results are as follows Figure 19As shown, strain Y309 had a strong inhibitory effect on the mycelial growth of all 12 tested plant pathogenic fungi. Among them, the strongest inhibitory effect was on Rhizoctonia graminearum, Pseudomonas aeruginosa, and Aspergillus niger, with inhibition rates exceeding 70%, at 80.9%, 76.1%, and 71.1%, respectively. The inhibition rates against Colletotrichum destructans, Fusarium oxysporum, Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, Fusarium pseudograminearum, and Botrytis cinerea ranged from 60.6% to 69.0%, at 69.0%, 68.0%, 67.9%, 66.0%, 65.4%, 64.6%, 63.9%, and 60.6%, respectively. The inhibition rate against Septoria celeriac was the lowest, at only 50.2%. This indicates that strain Y309 has a strong broad-spectrum antifungal activity.

[0115] (2) Antibacterial ability of strain Y309 against pathogenic bacteria The double-layer plate method was used to determine the Y309 strain's resistance to the pathogenic variants of Pseudomonas syringae ( Pseudomonas syringae pv. Lachrymans )、Xanthomonas campestris pv. capsici ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Agrobacterium tumefaciens ( Agrobacterium vitis )、Carrot Pectin Bacillus( Pectobacterium carotovorum ), Bacillus cereus ( Bacillus cereus ) Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ),salmonella( Salmonella sp.) and other 10 pathogenic bacteria. Y309 strain was picked with a sterile toothpick and inoculated into the center of a PDA plate. After culturing in a 28°C incubator for 5 days, 5 mL of chloroform was used to kill the strain by inverted fumigation. After the chloroform was completely evaporated, a solution containing 2% OD 600 = 0.8, and placed in a 28°C incubator for 24 h. The diameter of the transparent inhibition zone was measured by the cross method and calculated according to Formula 3).

[0116] The results are as follows Figure 20As shown in the results, the Y309 strain had the strongest inhibitory effect on Pseudomonas syringae pv. lacrimalis and Bacillus cereus, with inhibition zone diameters exceeding 30 mm, which were 34.75 mm and 32.17 mm, respectively. The inhibition zone diameters against Staphylococcus aureus, Salmonella, Xanthomonas campestris pv. capsici, Ralstonia solanacearum, and Xanthomonas citri subsp. citri were 21.71 mm, 21.44 mm, 20.58 mm, 13.51 mm, and 5.23 mm, respectively. However, it had no inhibitory effect on Escherichia coli, Agrobacterium tumefaciens, and Pectobacterium carotovora.

[0117] Example 5 Growth-promoting effect of strain Y309 on wheat The wheat variety used was Jimai 22 (bred by the Shandong Academy of Agricultural Sciences). Y309 strain was inoculated into liquid PD medium and shake-cultured at 28°C for 7 days to prepare a Y309 bacterial suspension. Wheat seeds were surface-sterilized by soaking in 75% alcohol for 30 seconds. The seeds were rinsed 2–3 times with sterile water to remove residual alcohol. The seeds were then soaked in the original suspension and in 100-, 500-, and 1000-fold dilutions in the dark for 3 hours. Germination was then accelerated in germination boxes at 28°C in the dark, with 100 seeds placed in each box to minimize variation. Each treatment was replicated four times, with sterile water treatment serving as the control. Seed germination potential and germination rate were measured on the third and fifth days, respectively. Germination criteria were defined as "radicle length equal to or greater than the seed length, and the embryo exceeding half the seed length." Shoot length, root length, dry weight, and fresh weight of individual seedlings were measured on the fifth day. Seeds with obvious necrosis were removed promptly to avoid contamination.

[0118] The results are as follows Figure 21As shown, fermentation broth of strain Y309 at different dilution ratios had varying degrees of effect on wheat growth. With increasing dilution ratios, the fermentation broth's effect on wheat seed germination and seedling growth significantly increased. Soaking wheat seeds with a 1000-fold dilution of the fermentation broth had the most significant growth-promoting effect. The group treated with the original fermentation broth had the lowest germination potential, shoot length, and root length, decreasing by 52.7%, 16.4%, and 27.9%, respectively, compared to the control. This suggests that the original fermentation broth of strain Y309 significantly inhibited wheat seed germination and seedling growth, producing high concentrations of plant growth substances that inhibited seed growth. After soaking wheat seeds in a 500-fold dilution of the fermentation broth for three days, the germination potential of the seeds reached approximately 75%, significantly higher than in the other treatments. The wheat germination potential in the 1000-fold diluted fermentation liquid treatment group reached 75%, with shoot and root lengths reaching 95.07 mm and 91.25 mm, respectively, and a fresh weight of 0.19 g. Compared with the control group not inoculated with the fermentation liquid, all growth parameters increased significantly, with wheat germination potential, shoot length, root length, and fresh weight increasing by 11.7%, 27.5%, 48.5%, and 26.7%, respectively. Treatment with the 500-fold diluted fermentation liquid had a certain growth-promoting effect on wheat germination potential and root length, while shoot length and fresh weight showed no significant differences from the control group.

[0119] Example 6 Determination of the control effect of strain Y309 on wheat sheath blight on plates The pathogen of wheat sheath blight was Rhizoctonia graminearum ( Rhizoctonia solani ), the wheat varieties were the same as those in Example 5. The Y309 strain suspension was prepared using the same method as in Example 5. After surface disinfection, the wheat seeds were soaked in sterile water for 3 hours and then placed in a petri dish at 28°C for 48 hours to moisturize and accelerate germination. Wheat seeds of uniform size and consistent whitening were selected and placed on plates infested with Rhizoctonia graminearum, with 10 seeds placed per plate. A layer of sterile vermiculite was then poured over the plates. Using the root irrigation method, 100 µL of sterile water, the original bacterial suspension, and 5- and 10-fold dilutions of the bacterial suspension were injected into the roots of the seeds. A 1000-fold dilution of 24% Validamycin A solution was used as a positive control. The seeds were cultured in a greenhouse at 25°C, watered daily to keep the vermiculite moist. After 14 days, the wheat was removed with its roots, and the degree of browning of the roots and leaf sheaths was observed. The disease grading standard for wheat sheath blight was based on the following statistical criteria. Formulas 6) to 8) were used to calculate the disease grade index and control efficacy of each treatment.

[0120] Grade 0: asymptomatic; Level 1: Leaf sheath turns brown; Level 2: There are obvious sheath blight spots on the outside of the leaf sheath, with a diameter of less than 0.5 cm; Level 3: One to several lesions on the outside of the leaf sheath, with a diameter of >0.5 cm; Level 4: There are lesions inside the leaf sheath; Level 5: There are spots on the stems or the plants are dead.

[0121] The results are as follows Figure 22 As shown, the fermentation solution of the Y309 strain effectively controls wheat sheath blight. The wheat in the control group inoculated only with Rhizoctonia graminearum suffered severe disease, with noticeable browning at the base of the stems, and an incidence rate and disease index of 80.0% and 36.00, respectively. Treatment with the fermentation solution of the strain and with Jinggangmycin significantly reduced the incidence and disease index of wheat sheath blight, with efficacy rates of 52.8% and 50.0%, respectively, showing no significant difference. In contrast, the fermentation solution of the Y309 strain, after a five-fold dilution, still exhibited some control efficacy against wheat sheath blight, with incidence and disease index reduced by 15.7% and 40.3%, respectively, compared to the control group, achieving a relative efficacy of 40.3%.

[0122] Example 7 Determination of the greenhouse efficacy of strain Y309 against tomato gray mold The pathogen of tomato gray mold was Botrytis cinerea ( Botrytis cinerea ), the tomato variety was selected from Fanteli (Shouguang Gedun Agricultural Technology Co., Ltd.). The preparation method of the Y309 strain suspension was the same as in Example 5.

[0123] Healthy and uniformly sized tomato leaves were picked from tomato plants that had grown to the six-leaf stage. They were washed 2-3 times with sterile water to remove surface impurities. After natural drying, they were immersed in sterile water, the original bacterial suspension, and 5-fold and 10-fold diluted bacterial suspensions for 10 s, respectively. After drying, a 5 mm Botrytis cinerea cake was inverted on the leaf surface. A 1000-fold dilution of 50% procymidone wettable powder was used as a positive control. The treated leaves were placed in a Petri dish lined with 4 layers of moist filter paper and incubated in the dark at 22°C for 3 days. Three replicates were set for each treatment, with 10 leaves in each replicate. After the control was fully diseased, the leaves were photographed under bright field and UV light (365 nm). The lesion area was measured using Image J. The control effect was calculated according to Formula 9): The results are as follows Figure 23 As shown in the results, the fermentation liquid of strain Y309 has a good control effect on tomato gray mold. The control effect of the fermentation liquid is slightly lower than that of procymidone, but the difference is not significant, both of which are above 65%. After being diluted 5 times, the fermentation liquid still has a certain control effect on tomato gray mold, with a control effect of 27.9%.

[0124] Example 8: Greenhouse efficacy of strain Y309 against pepper wilt The pathogen of pepper wilt was Fusarium oxysporum ( Fusarium oxysporum), the pepper variety used was Zhongjiao No. 6 (bred by the Beijing Academy of Agricultural Sciences). To prepare the fungus soil, Fusarium oxysporum was inoculated into a corn substrate and cultured at 22°C for approximately 10 days. After the mycelium had fully grown throughout the medium, the fungus-infested corn substrate was added at a 3% (w / w%) concentration with a soil substrate (nutrient soil and vermiculite mixed in a 1:1 volume ratio) to prepare the fungus soil. The preparation method for the Y309 strain suspension was the same as in Example 5.

[0125] After disinfection, pepper seeds were soaked in Y309 strain suspension, 500 times 50% carbendazim wettable powder and sterile water for 30 minutes before sowing. Four seeds were sown in each pot, and 10 replicates were used for each treatment. The water treatment without pathogenic bacteria was used as a control. 21 days after emergence, the peppers were pulled out with their roots and the disease condition of the plants was observed. The pepper wilt disease grading standard was based on the severity of vascular tissue discoloration. The disease grade index and prevention effect of each treatment were calculated according to the following statistics.

[0126] Level 0: Roots are healthy and asymptomatic; Level 1: The roots are slightly browned, with the browning area ≤ 33%; Level 2: Root rot occurs, with browning area ≤ 67%, and lesions appear at the base of the stem; Level 3: The root system is severely rotten, with browning area ≥ 67%, and the plant wilts or even dies.

[0127] The results are as follows Figure 24 As shown, the fermented liquid of strain Y309 effectively prevents and controls pepper wilt. Compared with healthy plants inoculated with no pathogenic bacteria, the treatment group infected with Fusarium oxysporum showed a 100% disease incidence rate, with significant dwarfing and root rot. However, the aboveground leaves showed no obvious signs of water shortage and chlorosis, with a disease index of 38.73. Both treatments with strain Y309 and carbendazim significantly reduced the incidence of pepper wilt, with disease indices dropping to 22.29 and 30.09, respectively. The control efficacy of strain Y309 reached 40.0%, significantly higher than that of the carbendazim-treated group, which only achieved a 22.3% efficacy. However, when various growth indicators of pepper plants were measured, the plant height and root length of the fermentation liquid treatment group were significantly higher than those of the sterile water and carbendazim treatment groups, but lower than the control. The fresh weight of a single plant had no significant difference from the carbendazim treatment group, and decreased by 38.0% compared with the CK treatment, indicating that the Y309 strain failed to completely alleviate the growth inhibitory effect of Fusarium oxysporum on tomatoes.

[0128] Example 9: Effect of strain Y309 on corn growth promotion in saline-alkali soil in Xinjiang This example set up two potted experiments to further verify the growth-promoting effect of strain Y309 on corn in saline-alkali soils in Xinjiang. The corn variety used was Jinchong No. 1 (Xinjiang Meiya Lianda Seed Co., Ltd.). The preparation method for the Y309 bacterial suspension was the same as in Example 5. Basic physical and chemical properties of saline-alkali soils and non-saline-alkali soils in Xinjiang are shown in Table 13. 1.5 kg of bacterial suspension was added to each pot for both soil types, and 50 mL of bacterial suspension was added. To maintain soil moisture, 100 mL of sterile water was added to the suspension. In the non-saline-alkali soil test, 2 g / kg of sodium chloride was added to the sterile water. The sample soil was spread evenly on a sheet of water-resistant kraft paper and sprayed evenly. After each layer was evenly sprinkled with the bacterial suspension, the soil was gently mixed, minimizing soil disturbance. The suspension was then added until all the bacterial suspension had been added. A fermentation medium without the bacterial suspension was sprayed and mixed as a blank control. After addition, 1.5 kg of soil was weighed and placed in an 18 cm diameter plastic pot. Three corn seeds with uniform germination were sown in each pot at a sowing depth of about 1 cm. Water was applied every two days after sowing with 100 mL of water. Thinning was started 3 days after emergence, leaving only two corn plants of uniform size in each pot. MS nutrient solution (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was added 10 days after thinning.

[0129] Table 13 Basic physical and chemical properties of different types of soil

[0130] Samples were collected starting 40 days after sowing to test the growth-promoting ability of the Y309 strain fermentation liquid on corn seedlings. A handheld chlorophyll meter (SPAD-502PIU) was used to measure the largest leaf from top to bottom three times, and the average SPAD value was used as the SPAD value of the corn plant. Growth indicators such as corn seedling height, stem diameter, aboveground fresh weight, aboveground dry weight, maximum leaf area, and chlorophyll were measured.

[0131] The results are shown in Table 14. Strain Y309 has a good growth-promoting effect on the aboveground part of corn in Xinjiang saline-alkali soil ( Figure 25 ), which can increase the height of corn seedlings by 11.81±0.03%, the stem diameter by 43.11±0.16%, the aboveground fresh weight by 41.57±0.13%, the aboveground dry weight by 42.16±0.32%, the leaf area by 3.15±0.05%, and the chlorophyll by 1.15±0.02%.

[0132] Table 14 Effect of strain Y309 on corn growth promotion in saline-alkali soil in Xinjiang

[0133] Note: Different lowercase letters indicate significant differences.

[0134] The results are shown in Table 15. Strain Y309 has a good growth-promoting effect on the aboveground and underground parts of corn in Xinjiang non-saline-alkali soil with added salt ( Figure 26 ), which can increase the height of corn seedlings by 26.52±0.09%, the stem diameter by 26.78±0.12%, the aboveground fresh weight by 0.55±0.14%, the underground fresh weight by 19.69±0.16%, the aboveground dry weight by 52.73±0.30%, the leaf area by 13.03±0.19%, and the chlorophyll by 9.95±0.02% (Table 15).

[0135] Table 15 Effect of strain Y309 on corn growth promotion in Xinjiang non-saline soil with added salt

[0136] Note: Different lowercase letters indicate significant differences.

[0137] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Streptomyces Streptomyces sp. Y309, deposit number GDMCC No: 64527.

2. A microbial agent, biological pesticide or biological fertilizer prepared from the Streptomyces according to claim 1, its fermentation liquid or a crude extract of the fermentation liquid.

3. A biocontrol agent or antibacterial agent prepared by the Streptomyces according to claim 1.

4. Any of the following uses of the Streptomyces or its bacterial agent according to claim 1: 1) Used to antagonize plant pathogenic fungi; 2) Used to antagonize plant pathogenic bacteria; 3) Used to prevent and treat pepper wilt; 4) Used to prevent and control wheat sheath blight; 5) Used to prevent and control tomato gray mold; 6) Used for the preparation of biological pesticides; 7) Used for preparing biofertilizer; 8) Used for plant growth promotion.

5. The use according to claim 4, characterized in that: The plant pathogenic fungi include Pseudomonas graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Fusarium moniliforme( Fusarium moniliforme )、Pseudomonas verticillasteroides( Fusarium verticillioides )、Botrytis cinerea( Botrytis cinerea ), Rhizoctonia graminearum ( Rhizoctonia cerealis )、Rhizoctonia solani( Rhizoctonia solani )、Sweet potato long beaked shell fungus( Ceratocystis fimbriata ) and Alternaria solani ( Alternanria solani )、Brown rot fungi( Monilia laxa ), destroy anthrax ( Colletotrichum destructivum )、Septoria celeriac( Septoria apiicola ).

6. The use according to claim 4, characterized in that: The plant pathogenic bacteria include Pseudomonas syringae pv. Pseudomonas syringae pv. Lachrymans )、Xanthomonas campestris pv. capsici ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella sp.).

7. The use according to claim 4, characterized in that: The plants mentioned in 8) include wheat and pepper.

8. Any of the following uses of the Streptomyces according to claim 1 and / or the antibacterial active substances produced therefrom: a) for the prevention of pathogens and plant diseases caused by pathogens; b) for inducing plants to enhance resistance to plant diseases caused by pathogenic bacteria; c) Used for the preparation of biocontrol agents, antimicrobial agents or plant immunity inducers.

9. The use according to claim 8, characterized in that: a) The pathogens include pathogenic fungi and pathogenic bacteria; wherein the pathogenic fungi include Pseudomonas graminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum )、Fusarium moniliforme( Fusarium moniliforme )、Pseudomonas verticillasteroides( Fusarium verticillioides )、Botrytis cinerea( Botrytis cinerea ), Rhizoctonia graminearum ( Rhizoctonia cerealis )、Rhizoctonia solani( Rhizoctonia solani )、Sweet potato long beaked shell fungus( Ceratocystis fimbriata ) and Alternaria solani ( Alternanria solani )、Brown rot fungi( Monilia laxa ), destroy anthrax ( Colletotrichum destructivum )、Septoria celeriac( Septoria apiicola ); The pathogenic bacteria include Pseudomonas syringae pityrosporum ( Pseudomonas syringae pv. Lachrymans )、Xanthomonas campestris pv. capsici ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella sp.); The plant diseases mentioned in b) include wheat sheath blight, tomato gray mold, and pepper wilt.

10. Any of the following uses of the Streptomyces according to claim 1: i) For the production of siderophore; ii) used to produce IAA; iii) used to dissolve organic phosphorus; iv) Used for hydrolysis of starch and cellulose; v) Used for coagulation and peptization of milk.

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