Streptomyces sp. Y309 and Its Application

Abstract

The present invention belongs to the field of microbial technology and discloses Streptomyces sp. Y309 and its application. The Streptomyces Streptomyces sp. Y309, with the preservation number GDMCC No: 64527, has a strong ability to synthesize secondary metabolites and can produce siderophores, IAA, etc. This strain also has the potential for plant growth promotion and disease control. Strain Y309 has strong antibacterial effects on 12 pathogenic fungi such as Fusarium oxysporum, Rhizoctonia cerealis, Monilinia fructicola, and Ceratocystis fimbriata, as well as 7 pathogenic bacteria such as Pseudomonas syringae pv. lachrymans and Bacillus cereus. It has good control effects on wheat sharp eyespot, tomato gray mold, and pepper fusarium wilt. The fermentation broth of strain Y309 can significantly promote the germination of wheat seeds and the growth of seedlings, and can improve the agronomic traits such as plant height, dry weight, and leaf area of maize seedlings in different saline-alkali soils, showing good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically relates to a Streptomyces sp. Y309 and its application. Background Art

[0002] Plant diseases caused by fungi seriously affect global food security. There are more than 19,000 pathogenic fungi that can cause global crop diseases, mainly concentrated in the phyla Ascomycota and Basidiomycota. Among them, Fusarium oxysporum, Rhizoctonia cerealis, and Botrytis cinerea are all world-wide pathogenic fungi due to their wide host range, high incidence rate, and serious harm, endangering various regions of the world.

[0003] The wilt disease caused by Fusarium oxysporum is a serious vascular system disease that can infect multiple crops such as soybeans, bananas, cotton, tomatoes, and peppers. Long-term continuous cropping, soil physical and chemical properties such as soil acidity and compaction, and greenhouse planting conditions such as high temperature and high humidity are all important conditions for promoting the occurrence and aggravation of wilt disease. Among them, the incidence rate of pepper wilt disease is generally 15% - 30%, and it occurs in all pepper planting areas. The sheath blight disease caused by Rhizoctonia cerealis is an important disease in the process of wheat production. The occurrence area of wheat sheath blight in China has been increasing year by year, and the incidence rate in severely diseased areas is as high as over 70%, resulting in a yield reduction of more than 30%. The gray mold disease caused by Botrytis cinerea is a common disease in tomato planting, which can infect the stems, leaves, flowers, fruits, etc. of plants, and the annual yield loss is as high as 20% - 50%.

[0004] The genus Streptomyces is an important resource microorganism with complex secondary metabolic capabilities. Although natural products derived from the genus Streptomyces only account for 39% of the currently isolated microbial secondary metabolites, they produce nearly 80% of the bioactive compounds. The genus Streptomyces can directly or indirectly improve the growth and development of plants by producing plant hormones, improving soil nutrients, regulating plant metabolism, and enhancing plant stress resistance. Due to the differences in their growth habitats, nutritional conditions, etc., Streptomyces strains from different sources also have diversity in natural product synthesis. The development of Streptomyces resources with excellent traits can effectively delay the harm caused by the abuse of chemical pesticides to the ecological environment and human health. Summary of the Invention

[0005] The object of the present invention is to provide a Streptomyces sp. Y309 and its application. The strain Y309 has a strong ability to synthesize secondary metabolites and broad-spectrum antibacterial activity, and has a strong inhibitory effect on common pathogenic fungi and bacteria, especially its application in the biological control of plant diseases such as pepper wilt disease, wheat sheath blight disease, and tomato gray mold disease.

[0006] To achieve the object of the present invention, in a first aspect, the present invention provides a Streptomyces strain with biocontrol function. Streptomyces sp. Y309, which is a new species in the genus Streptomyces, and its taxonomic name is Streptomyces sp., this strain was isolated from Aksu Prefecture, Xinjiang Uygur Autonomous Region in 2021, and has now been deposited in the Guangdong Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, 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, biological pesticide or biological fertilizer 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 from Streptomyces Y309.

[0010] In a fifth aspect, the present invention provides any one of the following applications of Streptomyces Y309 or its microbial agent:

[0011] 1) For antagonizing plant pathogenic fungi;

[0012] 2) For antagonizing plant pathogenic bacteria;

[0013] 3) For controlling pepper wilt;

[0014] 4) For controlling wheat sharp eyespot;

[0015] 5) For controlling tomato gray mold;

[0016] 6) For preparing biological pesticides;

[0017] 7) For preparing biological fertilizers;

[0018] 8) For promoting plant growth.

[0019] The plant pathogenic fungi described in the present invention include but are not limited to Fusarium pseudograminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium moniliforme ( Fusarium moniliforme ), Fusarium verticillioides ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia cerealis ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani), Ceratocystis fimbriata Ceratocystis fimbriata ), Alternaria solani Alternanria solani ), Phellinus noxius Monilia laxa ), Colletotrichum destructivum Colletotrichum destructivum ), Septoria apii Septoria apiicola ).

[0020] The pathogenic bacteria include, but are not limited to, Pseudomonas syringae pv. lachrymans Pseudomonas Syringae pv. Lachrymans ), Xanthomonas campestris pv. vesicatoria 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 sp. Salmonella sp.).

[0021] The plants described in the foregoing application 8) include, but are not limited to, wheat and chili peppers.

[0022] In a sixth aspect, the present invention provides any one of the following applications of Streptomyces sp. Y309 and / or the antibacterial active substance produced by it:

[0023] a) For combating pathogenic bacteria and plant diseases caused by pathogenic bacteria;

[0024] b) For inducing plants to enhance their resistance to plant diseases caused by pathogenic bacteria;

[0025] c) For preparing biocontrol agents, antibacterial agents or plant immune inducers.

[0026] Furthermore, the pathogenic bacteria include pathogenic fungi and pathogenic bacteria, such as the aforementioned plant pathogenic fungi and plant pathogenic bacteria.

[0027] Furthermore, the plant diseases include, but are not limited to, chili wilt, wheat sheath blight, and tomato gray mold.

[0028] In a seventh aspect, the present invention provides any one of the following applications of Streptomyces sp. Y309:

[0029] i) For producing siderophores;

[0030] ii) For producing IAA;

[0031] iii) For dissolving organic phosphorus;

[0032] iv) For hydrolyzing starch and cellulose;

[0033] v) For solidifying and peptizing milk.

[0034] The present invention has carried out polyphasic identification on the antagonistic strain Streptomyces Y309 with broad-spectrum antibacterial activity isolated from the desert environment of Aksu, Xinjiang. This bacterium has potential biocontrol application value and good development and application prospects.

[0035] The fermentation medium for fermenting and culturing the Streptomyces Y309 of the present invention is: 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.

[0036] The present invention also relates to the stability and basic characteristics of the antifungal active substance of Streptomyces Y309. The antibacterial substance is stable at pH 4 - 9, after being treated at 60 °C for 1 h and irradiated with ultraviolet light for 40 min. The inhibition rates of the n-butanol extraction phase in the strain fermentation broth and the fermentation broth against Fusarium oxysporum are 61.2% and 63.4% respectively, with no significant difference, and the crude protein extract and the ethyl acetate extraction phase have weak activities.

[0037] Strain Y309 has strong inhibitory effects on the mycelial growth of 12 tested plant pathogenic fungi. Among them, the inhibitory effects on Rhizoctonia cerealis, Monilinia fructicola, and Ceratocystis fimbriata are the strongest, with inhibition rates of 80.9%, 76.1%, and 71.1% respectively. The inhibition rates on the remaining pathogenic fungi, such as Pseudograminearum, Fusarium oxysporum, Fusarium moniliforme, Verticillium dahliae, Botrytis cinerea, Rhizoctonia solani, Alternaria solani, and Colletotrichum destructivum, all exceed 60%. The inhibition rate on Septoria apii is the lowest, only 50.2%. Strain Y309 has the strongest inhibitory effects on Pseudomonas syringae pv. lachrymans and Bacillus cereus, with the inhibition zone diameters exceeding 30 mm, which are 34.75 mm and 32.17 mm respectively. The inhibition zone diameters for Xanthomonas campestris pv. vesicatoria, Ralstonia solanacearum, Xanthomonas citri subsp. citri, Staphylococcus aureus, and Salmonella are in the range of 5.23 - 22.80 mm, but there is no antibacterial effect on Escherichia coli, Agrobacterium tumefaciens, and Pectobacterium carotovorum. The results of greenhouse pot experiments show that the control effects of strain Y309 on the basal disease wheat sharp eyespot, the leaf disease tomato gray mold, and the root disease pepper wilt reach 52.8%, 65.1%, and 40.0% respectively. The fermentation broth of strain Y309 can significantly promote the growth of wheat, and the seed germination and the dry weight per plant of the seedlings of wheat are increased by 11.7% and 23.9% respectively. Strain Y309 has great potential in the prevention and control of plant diseases and good application prospects in biological pesticides and antibacterial agents.

[0038] The Streptomyces sp. 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 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, and distilled water 1000 mL, the fermentation broth has the strongest activity against Fusarium oxysporum. Its active substances are stable at pH 4-9, after being treated at 60 °C for 1 h and irradiated with ultraviolet light for 40 min, indicating that this strain has the potential for plant growth promotion and disease control. Strain Y309 has strong antibacterial effects against 12 pathogenic fungi such as Fusarium oxysporum, Rhizoctonia cerealis, Phaeosclera sp., and Ceratocystis fimbriata, and 7 pathogenic bacteria such as Pseudomonas syringae pv. lachrymans and Bacillus cereus. It has good control effects on wheat sharp eyespot, tomato gray mold, and pepper wilt. The fermentation broth of strain Y309 can significantly promote the germination of wheat seeds and the growth of seedlings, and can improve agronomic traits such as the plant height, dry weight, and leaf area of maize seedlings in different saline-alkali soils, indicating that this strain has good prospects for development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a phylogenetic tree constructed based on the 16S rRNA gene of strain Y309 of the present invention and the similar type strain of the genus Streptomyces in the preferred embodiment.

[0040] Figure 2 It is a phylogenetic tree based on the housekeeping genes of strain Y309 of the present invention and the similar type strain of the genus Streptomyces in the preferred embodiment.

[0041] Figure 3 It is a phylogenetic tree based on the whole genome sequence of strain Y309 of the present invention and the similar type strain of the genus Streptomyces in the preferred embodiment.

[0042] Figure 4 It is the morphological characteristics of strain Y309 in the preferred embodiment of the present invention. (a) Culture characteristics of strain Y309 (left: reverse side of the colony; right: front side of the colony); (b) Optical microscope photograph of strain Y309; (c) Scanning electron microscope photograph of strain Y309.

[0043] Figure 5 It is the physiological and biochemical characteristics of strain Y309 in the preferred embodiment of the present invention.

[0044] Figure 6 It is the influence of different media on the growth and antibacterial effect of strain Y309 in the preferred embodiment of the present invention. (a) Antibacterial rate of different fermentation media; (b) Colonies of Fusarium oxysporum at 1% addition amount of different fermentation media; (c) Antibacterial rate at 1% addition amount of different fermentation media; (d) Biomass of each treatment group at 1% addition amount of different fermentation media.

[0045] Figure 7 Carbon source optimization of strain Y309 in the preferred embodiment of the present invention. ①-⑨ are lactose, galactose, soluble starch, sucrose, glycerol, fructose, maltose, ISP2, and Natamycin in sequence.

[0046] Figure 8 Nitrogen source optimization of strain Y309 in the preferred embodiment of the present invention. ①-⑨ are soybean meal extract, peptone, soy peptone, tryptone, beef extract powder, ammonium sulfate, ammonium nitrate, ISP2, and Natamycin in sequence.

[0047] Fig. 9 Trace element optimization of strain Y309 in the preferred embodiment of the present invention.

[0048] Fig.10 Influence factor optimization of strain Y309 in the preferred embodiment of the present invention. ①-⑥ are xylose, methionine, cysteine, tyrosine, ISP2, and Natamycin in sequence.

[0049] Fig.11 Response surface optimization model verification of strain Y309 in the preferred embodiment of the present invention. ①-③ are unoptimized ISP2 medium (ISP), CCD model predicted optimal medium (UISP), and Natamycin in sequence.

[0050] Fig.12 Antibacterial concentration gradient of the fermentation broth of strain Y309 in the preferred embodiment of the present invention.

[0051] Fig.13 Antibacterial ability of the fermentation broth of strain Y309 under different acid-base treatment conditions in the preferred embodiment of the present invention.

[0052] Fig.14 Antibacterial ability of the fermentation broth of strain Y309 under different temperature treatment conditions in the preferred embodiment of the present invention.

[0053] Fig.15 Antibacterial ability of the fermentation broth of strain Y309 under different ultraviolet irradiation times in the preferred embodiment of the present invention.

[0054] Fig.16 Antibacterial ability of the crude protein extract of strain Y309 in the preferred embodiment of the present invention.

[0055] Fig.17 Antibacterial ability of the crude lipopeptide extract of strain Y309 in the preferred embodiment of the present invention.

[0056] Fig.18Antibacterial ability of the organic extraction phase and aqueous phase of strain Y309 in the preferred embodiment of the present invention. Figures a - e show the colony diameters of Fusarium oxysporum produced by adding the extraction phase / aqueous phase concentrated 20 times to the PDA plate; among them, "No.1" represents the first extraction with an organic solvent; "No.2" represents the second extraction with an organic solvent; "No.3" represents the third extraction with an organic solvent; "No.4" represents the fourth extraction with an organic solvent.

[0057] Fig.19 Antagonism of strain Y309 against pathogenic fungi in the preferred embodiment of the present invention.

[0058] Fig. 20 Antagonism of strain Y309 against pathogenic bacteria in the preferred embodiment of the present invention.

[0059] Fig.21 Growth promotion effect of strain Y309 on wheat in the preferred embodiment of the present invention. "1×" represents the original sterile fermentation broth; "100×" represents the sterile fermentation broth diluted 100 times; "500×" represents the sterile fermentation broth diluted 500 times; "1000×" represents the sterile fermentation broth diluted 1000 times.

[0060] Fig. 22 Control ability of strain Y309 against wheat sheath blight in the preferred embodiment of the present invention. "1×" represents the original sterile fermentation broth; "5×" represents the sterile fermentation broth diluted 5 times.

[0061] Fig.23 Control ability of strain Y309 against tomato gray mold in the preferred embodiment of the present invention. "1×" represents the original sterile fermentation broth; "5×" represents the sterile fermentation broth diluted 5 times.

[0062] Fig.24 Control ability of strain Y309 against pepper fusarium wilt in the preferred embodiment of the present invention.

[0063] Fig.25 Growth promotion effect of strain 309 on corn in Xinjiang saline - alkali soil in the preferred embodiment of the present invention.

[0064] Fig.26 Growth promotion effect of strain Y309 on corn in Xinjiang non - saline - alkali soil with added salt in the preferred embodiment of the present invention. Detailed implementation manners

[0065] The following examples are used to illustrate the present invention, but are not used 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.

[0066] The test bacteria involved in the following examples are as follows, all from China Agricultural University:

[0067] Pathogenic fungi: Fusarium pseudograminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium moniliforme ( Fusarium moniliforme ), Fusarium verticillioides ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia cerealis ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani ), Ceratocystis fimbriata ( Ceratocystis fimbriata ), Alternaria solani ( Alternanria solani ), brown rot pathogen ( Monilia laxa ), Colletotrichum destructivum ( Colletotrichum destructivum ), Septoria apii ( Septoria apiicola ).

[0068] Pathogenic bacteria: Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. Lachrymans ), Xanthomonas campestris pv. vesicatoria ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Agrobacterium tumefaciens ( Agrobacterium vitis ), Pectobacterium carotovorum ( Pectobacterium carotovorum ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Salmonella sp. ( Salmonella ).

[0069] Example 1 Identification of Strain Y309

[0070] 1. Isolation and purification of strain Y309

[0071] In 2021, soil samples were collected from Aksu Prefecture, Xinjiang Uygur Autonomous Region. 10 g of soil samples were weighed and added to a conical flask containing 100 mL of sterile normal saline. After standing for 20 min, the mixture was shaken on a shaker at 28 °C and 150 rpm for 30 min, then allowed to stand for 10 min. 100 µL of the supernatant was pipetted and added to a 1 mL centrifuge tube containing 900 µL of sterile normal saline for dilution, and then diluted successively by 10 2 , 10 3 , 10 4times. 100 µL of the above soil suspension was respectively spread on the Gause's No. 1 medium (soluble starch 20 g, potassium nitrate 1 g, dipotassium hydrogen phosphate 0.5 g, sodium chloride 0.5 g, magnesium sulfate heptahydrate 0.5 g, ferrous sulfate heptahydrate 0.01 g, distilled water 1000 mL, pH 7.2 - 7.4) containing 0.01% potassium dichromate. After culturing at 28 °C for 5 d, single bacterial colonies were picked with a sterile toothpick, purified by streaking, and stored for later use.

[0072] 2. Identification of Strain Y309

[0073] The strain Y309 was identified and described by using a polyphasic approach including molecular biology identification and phenotypic characteristics observation, and it was proved that this strain belongs to a potential new species in the genus Streptomyces. The details are as follows:

[0074] (1) 16S rRNA and housekeeping gene sequence analysis:

[0075] The genomic DNA of actinomycetes was extracted using a rapid bacterial genomic DNA extraction kit. Using the genomic DNA of strain Y309 as a template, the 16S rRNA (SEQ ID NO:1) of the strain and atpD , ikB , recA , rpoB , trpB (SEQ ID NO:2 - 6) and other 5 housekeeping genes were amplified by PCR. The amplification reaction system and conditions are shown in Tables 2 and 3. The sequenced and assembled sequences were subjected to online similarity analysis and alignment with all known gene sequences in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Subsequently, the Neighbor - Joining Method (NJ) in MEGA 11 software was used to construct the phylogenetic trees of 16S rRNA and housekeeping genes atpD - ikB - recA - rpoB - trpB in series to determine the phylogenetic relationship between strain Y309 and related strains.

[0076] Table 1 PCR primers used in this experiment

[0077]

[0078] Table 2 PCR reaction system

[0079]

[0080] Table 3 PCR reaction conditions

[0081]

[0082] The results are as Figure 1 、 Figure 2 shown. Strain Y309 has the closest phylogenetic relationship with S. aureoverticillatus JCM4347 T and the homology can reach 99.89%. However, the sequence similarity of the 5 housekeeping genes is between 94.30% and 97.86%. Among them, ikB the gene sequence similarity is 96.57%, which is lower than the recommended threshold of 98.5% for the division of new species. It is speculated that strain Y309 may be a potential new species of the genus Streptomyces.

[0083] (2)Whole-genome sequence analysis

[0084] Strain Y309 was sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for whole-genome sequencing using a sequencing method that combines second-generation Illumina and third-generation Pacbio. The online platforms JSpecies Web Server (http: / / jspecies.ribohost.com / jspeciesws / ) and Type(Strain)Genome Server (TYGS, https: / / tygs.dsmz.de / ) were used to compare strain Y309 with all known strains in the database, calculate the average nucleotide identity (ANI) and DNA-DNA hybridization value (DDH) between the two strains, and construct a phylogenetic evolution tree of the whole-genome sequence.

[0085] The results are as Figure 3 shown. The whole-genome size of strain Y309 is 10.05 Mb, and the genomic (G+C) mol% content is 71.75%. It consists of 1 chromosome and 3 plasmids. Among them, the size of the linear chromosome is 9.35 Mb, and 8410 coding genes are predicted, accounting for 85.7% of the whole genome. The phylogenetic evolution tree divides this bacterium and S. aureoverticillatus JCM4347 T into one branch ( Figure 3 a in Figure 3 ), but the dDDH and ANIb values between strain Y309 and the similar reference strains are between 22.6% and 45.1%, 77.4% and 91.8% ( S. aureoverticillatus b in TThe dDDH and ANIb values between them were 45.1% and 91.8% respectively, both lower than the thresholds (70%, 95%) for new species division, proving that Streptomyces sp. Y309 is a new species in the genus Streptomyces.

[0086] (3)Phenotypic characteristic analysis

[0087] The strain was inoculated on 7 ISP media, NA medium and Czapek medium in the International Streptomyces Project, and cultured at 28 °C for 21 d to observe characteristics such as substrate mycelium, aerial mycelium and soluble pigment. After the strain was inoculated on ISP2 medium and cultured at 28 °C for 14 d, slides were prepared and observed under an optical microscope (BX53F, Olympus, Japan) and an electron microscope (SU8100, Hitachi, Japan) for characteristics such as mycelium morphology, spore chain morphology and spore surface morphology.

[0088] The growth of strain Y309 was detected on ISP2 solid medium with good growth of the strain at different temperatures (4, 10, 20, 28, 37, 45, 50, 55 °C) for 7 d. The strain was inoculated on ISP2 medium with different pH values (4.0 - 12.0) and salt concentrations (0 - 10%, w / v%) and cultured at 28 °C for 7 d to detect the tolerance of the strain to pH and salt.

[0089] Detection of the IAA production ability of the strain: Streptomyces Y309 strain was streaked on a PDA plate for activation, and after culturing in a 28 °C constant temperature incubator for 7 d, a single colony disc with a diameter of 5 mm was punched and inoculated into liquid PD medium, cultured at 28 °C and 150 rpm for 7 days. After 7 days, the bacterial liquid was taken out, centrifuged at 12000 rpm for 5 min, and the content of IAA in the bacterial liquid was determined by the Salkowkin colorimetric method, with 3 replicates. The results showed that the amount of IAA produced by Y309 was 7.33 ± 0.02 mg / L.

[0090] For other physiological and biochemical characteristics, such as starch hydrolysis, gelatin liquefaction, nitrate reduction and other experiments, they were carried out according to the relevant operations in the "Manual for Systematic Identification of Common Bacteria". Using the bacterial identification version Biolog Gen Ⅲ, it was placed in a 28 °C incubator and incubated for 48 h. During the incubation period, the microplate was read multiple times using an automatic microbial rapid identification instrument (GEN Ⅲ, USA) to detect the utilization of 71 carbon sources such as dextrin and maltose by strain Y309 and the sensitivity to 23 chemical substances such as sodium lactate and lincomycin. Using the API ZYM kit to determine the activities of 19 enzymes such as alkaline phosphatase and esterase of the strain.

[0091] The morphological characteristics of strain Y309 on different culture media are shown in Figure 4For a in it, the results showed that strain Y309 grew well on ISP2, ISP3, ISP4, ISP7 and Czapek's medium, grew poorly on ISP1, ISP5, ISP6 and NA medium, and no soluble pigment was observed on all 9 media. In addition, the colony morphology, color of substrate mycelium and aerial mycelium, and sporulation of strain Y309 were different on different media, and the specific characteristics are shown in Table 4. When observing strain Y309 under the microscope, typical Streptomyces characteristics were found, with well-developed branched mycelia, long-chain-shaped spore chains, cylindrical spores, 0.8 - 1.1 µm, smooth surface, and no special structures such as spiny or scaly structures ( Figure 4 , b and c).

[0092] Table 4 Morphological characteristics of strain Y309 on different media

[0093]

[0094] Strain Y309 can grow within the temperature tolerance range of 15 - 45 °C, pH tolerance range of 6 - 11, and NaCl tolerance range of 0% - 8%. Strain Y309 can produce siderophores, IAA, and dissolve organic phosphorus, but cannot produce H2S; it can hydrolyze starch and cellulose, coagulate and peptize milk, but cannot hydrolyze aesculin; in addition, the strain was positive for urease, lipase, protease, oxidase, and catalase tests, and negative for citrate utilization, nitrate reduction, gelatin liquefaction, MR, VP, etc. ( Figure 5 ).

[0095] The results of API ZYM enzyme activity detection showed that the bacterium had enzyme activities such as alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, N-acetyl-glucosaminidase, α-mannosidase, and α-fucosidase, and did not have enzyme activities such as esterase (C4), lipase (C8), lipase (C14), and chymotrypsin.

[0096] The results of Biolog Gen Ⅲ showed that the Y309 strain could utilize trehalose, gentianose, turanose, lactose, glucose, mannose, melibiose, fructose, galactose, rhamnose, glycerol, etc. as the sole carbon source, but could not utilize maltose, cellobiose, raffinose, salicin, sucrose, mannitol, sorbitol, inositol, and adenosine, etc.; it could utilize nitrogen sources such as L-alanine, L-arginine, L-glutamic acid, and L-serine, but could not utilize nitrogen sources such as L-aspartic acid, D-serine, and D-aspartic acid, etc.; in addition, the strain could 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; it was sensitive to substances such as vancomycin, sodium butyrate, and minocycline, but could grow in the presence of substances such as sodium lactate, rifamycin, nalidixic acid, and aztreonam.

[0097] The strain Y309 in the present invention and the closely related model strains S. aureoverticillatus JCM4347 T There were significant differences in colony colors among 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 model strains (Table 5).

[0098] Table 5 Differential characteristics between strain Y309 and similar strains of Streptomyces

[0099]

[0100] Example 2 Fermentation conditions of strain Y309

[0101] The present invention provides a fermentation culture method suitable for high-yield antibacterial substances of Y309 strain. The optimized conditions include carbon source, nitrogen source, trace elements and influencing factors, the antibacterial activity of fermentation liquid against Fusarium oxysporum is used as the determination index, and the optimal culture medium is predicted by central combination experimental design.

[0102] (1) Preparation of seed solution

[0103] The 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 puncher and inoculated into 150 mL of liquid PD medium. The culture was carried out at 28 °C and 150 rpm for 5 days to prepare the seed solution.

[0104] (2) Fermentation conditions

[0105] 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.

[0106] (3)Growth determination of strain Y309

[0107] The growth of the strain was characterized by the dry weight of the mycelium. The cultured fermentation broth was centrifuged at 10,000 rpm for 10 min. The cell precipitate was rinsed 2 - 3 times with sterile water and then placed in an oven at 60 °C to dry to a 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.

[0108] (4)Activity determination of the fermentation broth of strain Y309

[0109] The centrifuged fermentation supernatant was filtered and sterilized through sterile filter membranes with pore sizes of 0.45 µm and 0.22 µm respectively, and the sterile fermentation broth was stored in a 4 °C refrigerator in the dark. Using Fusarium oxysporum as the target fungus, the growth inhibitory effect of the fermentation broth on Fusarium oxysporum was determined by the drug-containing plate method and the inhibition zone method.

[0110] The drug-containing plate method was used to determine the antibacterial activity. The sterile fermentation broth was added to the PDA medium that had been pre-melted and cooled to about 50 °C, gently shaken until evenly mixed, and then poured into a petri dish with a diameter of 60 mm to prepare a drug-containing plate with a final concentration of the fermentation broth of 1% - 5%. A 5 mm pathogen fungal cake was inoculated in the center of the plate, and it was cultured at a constant temperature of 28 °C for 3 d. The colony diameter was measured by the cross method, and the inhibition rate was calculated according to formula (1). The size of the inhibition rate characterized the strength of the antibacterial activity and the amount of antibacterial substances.

[0111]

[0112] The inhibition zone method was used to determine the antibacterial activity. 200 µL of a 1.0×10 6 cfu / mL spore suspension was added to the 130 mm × 130 mm PDA medium and spread evenly using a sterile glass bead. Sterile holes with a diameter of 7 mm were punched evenly on the plate, and the agar blocks were picked out. 100 µL of the sterile fermentation broth was added to the holes. The medium was placed in a 4 °C refrigerator overnight. After the liquid in the holes was completely absorbed, it was transferred to a 28 °C incubator for 36 h. The diameter of the transparent zone was measured by the cross method, and the diameter of the inhibition zone was calculated according to formula (2), using natamycin at 30 µg / mL as a correction.

[0113]

[0114] (5)Medium optimization

[0115] The drug-containing plate method was used to determine the growth inhibitory effect of 6 different fermentation media, such as ISP2, YM, YMG, T6, Czapek-Dox medium, and Gao's No. 1 medium, on Fusarium oxysporum, and the most suitable medium for the strain to produce antibacterial substances was screened. The medium formulations are as follows:

[0116] PDB medium: 200.0 g of potatoes, 20.0 g of glucose, 1000 mL of distilled water, natural pH.

[0117] ISP2 medium (Yeast extract-malt extract agar): 4.0 g of yeast extract, 4.0 g of glucose, 10.0 g of malt extract, 1000 mL of distilled water, pH 7.2 - 7.4.

[0118] Czapek medium (Czapek Dox Medium, hereinafter referred to as Czapek): 2.0 g of sodium nitrate, 0.5 g of potassium chloride, 30.0 g of sucrose, 1.0 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 1000 mL of distilled water, pH 7.0 - 7.4.

[0119] Gauze's Synthetic Medium No.1 (hereinafter referred to as Gauze's): 20.0 g of soluble starch, 1.0 g of potassium nitrate, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, 1000 mL of distilled water, pH 7.2 - 7.4.

[0120] YMG medium: 4.0 g of glucose, 4.0 g of yeast extract, 10.0 g of malt extract, 1000 mL of distilled water, pH 7.2.

[0121] YM medium: 3.0 g of yeast extract, 3.0 g of malt extract, 5.0 g of peptone, 10.0 g of glucose, 1000 mL of distilled water, pH 5.0 - 6.0.

[0122] T6 medium: 10.0 g of soluble starch, 10.0 g of glucose, 10.0 g of glycerol, 5.0 g of tryptone, 5.0 g of yeast extract, 1000 mL of distilled water, pH 7.0.

[0123] The results are as Figure 6 shown. Using ISP2 medium as the initial fermentation medium for strain Y309, subsequent optimization was carried out.

[0124] (6) Carbon source optimization

[0125] The malt extract was replaced with lactose, galactose, and soluble starch in equal amounts as carbon sources; the glucose was replaced with sucrose, glycerol, fructose, and maltose in equal amounts, and the other components were kept the same as the control. The antibacterial effect of the fermentation broth against Fusarium oxysporum was determined.

[0126] The results are as Figure 7As shown, different carbon sources have significant differences in the activity of the strain. When lactose and soluble starch are used to replace yeast extract powder in the initial medium, the antibacterial activity of the fermentation broth increases significantly. Considering the antibacterial activity and economic value of the strain comprehensively, soluble starch is selected for subsequent optimization research.

[0127] (7)Optimization of nitrogen source

[0128] On the basis of the optimal carbon source, the nitrogen sources are respectively replaced with soybean meal extract, peptone, soy peptone, tryptone, beef extract powder, (NH4)2SO4, and NaNO3 in equal amounts to replace yeast extract, and other components are kept consistent with the control. The antibacterial effect of the fermentation broth against Fusarium oxysporum is measured.

[0129] The results are as Figure 8 shown. When the initial yeast extract is used as the nitrogen source, the antibacterial activity is the highest, which is 22.79±0.78 mm. Therefore, the initial nitrogen source yeast extract is still selected for subsequent optimization research.

[0130] (8)Optimization of trace elements

[0131] Ten kinds of trace element salt ions (chloride salts): Fe(Ⅱ), Mn(Ⅱ), Cu(Ⅱ), Zn(Ⅱ), Co(Ⅱ), Ni(Ⅱ), V(Ⅲ), Se(Ⅳ), Mo(Ⅴ), and W(Ⅵ) are added externally to the medium. Concentration gradients of 1, 5, 10, 15, and 20 mg / L are set respectively. Without adding the above trace elements as the control group, the antibacterial effect of the fermentation broth against Fusarium oxysporum is measured.

[0132] The results are as Figure 9 shown. The addition of Cu(Ⅱ) significantly improves the antibacterial activity of the strain, and there is no significant difference among the addition amounts of 5, 10, and 15 mg / L of Cu(Ⅱ). Therefore, 5 mg / L of Cu(Ⅱ) is selected for subsequent optimization experiments.

[0133] (9)Optimization of influencing factors

[0134] On the basis of the optimal carbon source, nitrogen source, and trace elements, 100 mg / L of methionine, cysteine, tyrosine, and pentose xylose (5 g / L) that may affect the metabolic process are screened, and the antibacterial effect of the fermentation broth against Fusarium oxysporum is measured.

[0135] The results are as Figure 10 shown. Xylose is incorporated into the fermentation medium of the strain for subsequent optimization.

[0136] (10)Plackett-Burman experimental design

[0137] To determine the influence degree of the selected culture medium components, with the diameter of the antibacterial circle of the fermentation broth as the response value, Design-Expert 13.0 was used to design the PB experiment, and the 5-factor 2-level design is shown in Table 6.

[0138] Table 6 Factors and levels of Plackett-Burman experimental design

[0139]

[0140] The results of the PB experiment are shown in Table 7. The complete loss of the antibacterial activity of the fermentation broth of multiple components among them is when xylose is at the high "1" level, indicating that a xylose concentration of 8 g / L is not suitable for the growth and fermentation of the strain. Therefore, it is considered that the influence of the 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 P xylose (E) with < 0.0001 is the most important factor affecting the strain activity, followed by glucose ( P = 0.0254) and yeast extract ( P = 0.0281). The prediction curve shows that both yeast extract and xylose have an obvious negative effect on the response value, while the addition amount of glucose has a positive effect on the response value.

[0141] Table 7 Plackett-Burman experimental design scheme and results

[0142]

[0143] Table 8 Plackett-Burman experimental variance analysis

[0144]

[0145] (11) Steepest ascent experiment

[0146] According to the factors significantly affecting the antibacterial activity of strain Y309 screened by the Plackett-Burman experiment, to determine the positive and negative of the significant factor effects, set appropriate step sizes and climbing directions to make the addition amounts of the significant factors approach the optimal region, find the center point for the response surface model, and determine the optimal addition amounts of each significant factor.

[0147] The results are shown in Table 9. The levels of each factor corresponding to Experiment 3 can make the activity of strain Y309 reach the maximum value. Therefore, this addition amount is selected as the center point for the subsequent central composite experiment.

[0148] Table 9 Steepest ascent experimental design and results

[0149]

[0150] (12)Central composite design of culture medium components

[0151] Use Design-Expert 13.0 to design the Central Composite experiment. Each factor takes five levels of -2, -1, 0, 1, and 2, establish a multiple regression equation, and predict the optimal concentration values of each factor to provide a feasible solution for the strain with high antibacterial activity in liquid fermentation (Table 10).

[0152] Table 10 Factors and levels of Central Composite experimental design

[0153]

[0154] The central composite design (CCD) under the response surface method (RSM) determined the optimal values of the variables significantly affecting the antibacterial activity of the strain through 5 different levels and 20 experimental combinations. The experimental design and results are shown in Table 11. By performing a regression analysis on the response value results (Table 12), a non-linear regression equation for the influence of each factor on the diameter of the antibacterial circle can be obtained: 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 parameters of the model indicate the effectiveness of the model, indicating that the model can be used to predict the antibacterial activity of the fermentation broth of strain Y309. The optimized culture medium formula most suitable for the fermentation of strain Y309 predicted by the response surface optimization experiment is as follows: 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.

[0155] Table 11 Central composite experimental design and results

[0156]

[0157] Table 12 Analysis of variance of central composite experimental design

[0158]

[0159] 13) Fermentation optimization verification experiment

[0160] Perform shake flask fermentation verification on the optimal fermentation medium combination obtained from the response surface model, and set 3 replicates to verify the accuracy and feasibility of the response surface model.

[0161] The results are asFigure 11 As shown in the figure, the optimized culture medium significantly improved the antibacterial activity of the fermentation broth of strain Y309 against Fusarium oxysporum, with a 103.3% increase compared to the unoptimized level. The diameter of the inhibition zone reached 31.10 mm, and the difference was extremely significant. Moreover, the growth of the strain was more efficient, and the biomass could reach 0.85 g, which was 21.2% higher than that before optimization.

[0162] Example 3 Extraction and Stability Analysis of the Crude Extract of Strain Y309

[0163] (1) Preparation of the Fermentation Broth

[0164] The fermentation medium was the optimal medium predicted by response surface in Example 2, and the formula was as follows: 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 strain fermentation method was the same as that in Example 2.

[0165] (2) Determination of the Half - Inhibitory Concentration IC 50 of the Fermentation Broth against Fusarium oxysporum

[0166] Using the drug - containing plate method, the antibacterial rates of different fermentation broth concentrations from 0% to 5% were measured. The dose - effect curve of the sterile fermentation broth of the strain against Fusarium oxysporum was obtained by fitting the logistic equation using origin 2021, and IC 50 was calculated. The results are as Figure 12 shown. The half - inhibitory concentration IC 50 of the fermentation broth of strain Y309 against Fusarium oxysporum was 5.05 µL / mL.

[0167] (3) Acid - Base Stability of the Antibacterial Substance of Strain Y309

[0168] The pH of the fermentation broth was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, and 10 respectively with 1 mol / L HCl and NaOH solutions, and then adjusted back to the original state after standing at room temperature for 24 h. Using the untreated sterile fermentation broth as a control, the drug - containing plate method was used to measure the effect of different acid - base conditions on the antibacterial substance of strain Y309. The addition amount of the drug solution in the drug - containing plate was the calculated IC 50 concentration of 5.05 µL / mL. The results are as Figure 13 shown. The antibacterial substance in the fermentation broth had high acid - base stability. When the pH value was 4 - 9, its antibacterial activity had no significant difference from that of the original sterile fermentation broth. It indicated that the antibacterial substance synthesized by Y309 had strong acid - base stability.

[0169] (4) Temperature Stability of the Antibacterial Substance of Strain Y309

[0170] The fermentation broth was heated in a water bath at 40, 60, 80, and 100 °C for 1 h, and treated at 121 °C for 20 min using a high-pressure steam sterilizer. After the fermentation broth returned to room temperature, using the untreated sterile fermentation broth as a control, the disk diffusion method was used to determine the effect of different temperature treatments on the antibacterial substances of strain Y309. The added amount of the drug solution on the disk diffusion plate was the calculated IC 50 concentration of 5.05 μL / mL. The results are as Figure 14 shown. After the fermentation broth was treated at 40 - 60 °C for 1 h, there was no significant change in the antibacterial activity of the fermentation broth. After being treated at 80 °C for 1 h, about 80% of the activity of the fermentation broth could still be retained. This indicates that the antibacterial substances synthesized by Y309 have strong thermal tolerance stability.

[0171] (5)UV stability of the antibacterial substances of strain Y309

[0172] The sterile fermentation broth was placed open at a distance of 20 cm from a UV lamp (30 W) and irradiated for 20, 40, 60, 90, and 120 min respectively, and then taken out. Using the untreated sterile fermentation broth as a control, the disk diffusion method was used to determine the effect of different UV irradiation times on the antibacterial substances of strain Y309. The added amount of the drug solution on the disk diffusion plate was the calculated IC 50 concentration of 5.05 μL / mL. The results are as Figure 15 shown. After the fermentation broth was irradiated with UV for more than 40 min, the activity of the fermentation broth decreased significantly. As the UV irradiation time increased, the antibacterial activity of the fermentation broth continued to decline. After irradiation for 120 min, the activity of the fermentation broth was only about 40% of the original fermentation broth.

[0173] (6)Determination of the activity of the crude extract of water-soluble proteins of strain Y309

[0174] The water-soluble protein substances in the fermentation broth of strain Y309 were extracted using the ammonium sulfate precipitation method. The precipitate was dissolved in 0.01 M PBS, placed in a dialysis bag with a molecular weight cut-off of 3500 D for dialysis to remove salts, and then filtered and sterilized through a 0.22 μm sterile filter membrane to obtain the crude extract of water-soluble proteins. The protein content was determined using Coomassie Brilliant Blue G-250, and after gradient dilution, the protein content was 1, 5, 10, 15, and 20 times that of the original fermentation broth respectively. The disk diffusion method was used to determine the antibacterial activity of the solution. The added amount of the drug solution on the disk diffusion plate was the calculated IC 50 concentration of 5.05 μL / mL to detect whether the crude extract had antibacterial activity. The results are as Figure 16 shown. Although the protein substances in the fermentation broth of strain Y309 had certain activity against Fusarium oxysporum, indicating that Y309 synthesized protein antibacterial substances, they were not the only antibacterial substances.

[0175] (7)Activity determination of the crude extract of water-soluble lipopeptides from strain Y309

[0176] The lipopeptide substances in the fermentation broth of strain Y309 were extracted by acid precipitation and alcohol extraction method. The precipitate was ultrasonically assisted extracted with 10% methanol and filtered through a 0.22 µm sterile filter membrane to obtain the crude extract of water-soluble lipopeptides. After gradient dilution, solutions with volume concentrations of 1, 5, 10, 15, and 20 times that of the original fermentation broth were obtained. The antibacterial activity of the solutions was determined by the drug-containing plate method. The addition amount of the drug-containing plate solution was the calculated IC 50 concentration of 5.05 μL / mL to detect whether the crude extract had antibacterial activity. The results are as Figure 17 shown. The crude extracts of lipopeptides with different dilution multiples did not show antibacterial activity against Fusarium oxysporum. It shows that the antibacterial substance synthesized by Y309 is a non-lipopeptide substance.

[0177] (8)Activity determination of the crude extract of small molecules from strain Y309

[0178] Four consecutive extractions of different polar small molecule substances in the fermentation broth of strain Y309 were carried out using twice the volume of petroleum ether, toluene, ethyl acetate, and water-saturated n-butanol. The organic phases in the four extraction processes were collected and the final aqueous phase was preserved. The organic phase was evaporated to dryness at 60 °C using a rotary evaporator, and solutions with volume concentrations of 1, 5, 10, 15, and 20 times that of the original fermentation broth were obtained after gradient dilution with 10% methanol. The antibacterial activity of each phase was determined by the drug-containing plate method. The addition amount of the drug-containing plate solution was the IC 50 concentration of 5.05 µL / mL to detect whether the crude extract had antibacterial activity. The results are as Figure 18 shown. The active substances in the fermentation broth of strain Y309 belong to small molecule substances with medium polarity or even high polarity. The activity is concentrated in the n-butanol extraction phase, and the specific active substances need to be further studied for the crude substances in the n-butanol phase. It shows that the antibacterial substance synthesized by Y309 is easily soluble in n-butanol and has low solubility in petroleum ether, toluene, and ethyl acetate.

[0179] Example 4 Broad-spectrum antibacterial effect of strain Y309

[0180] (1)Antibacterial ability of strain Y309 against pathogenic fungi

[0181] The plate confrontation method was used to determine the antibacterial ability of strain Y309 against Fusarium pseudograminearum ( Fusarium pseudograminearum ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium moniliforme ( Fusarium moniliforme ), Fusarium verticillioides ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia cerealis ( Rhizoctonia cerealis ), Rhizoctonia solani ( Rhizoctonia solani ), Ceratocystis fimbriataCeratocystis fimbriata ), Alternaria solani Alternanria solani ), brown rot pathogen Monilia laxa ), Colletotrichum destructivum Colletotrichum destructivum ), Septoria apii Septoria apiicola ), etc. The growth inhibitory effects on 12 pathogenic fungi belonging to 8 genera were studied. Use a sterile toothpick to pick the Y309 strain and inoculate it at two positions 25 mm apart on the PDA plate. After incubating at 28 °C in an incubator for 5 days, inoculate a 5-mm pathogenic fungal cake into the center of two single colonies of the Y309 strain. Use the plate inoculated with only the pathogenic fungus as a control. After placing it in an incubator at 22 °C and incubating until the control is about to cover the plate, measure the diameter of the pathogenic fungus, and calculate the strain growth inhibition rate according to formula 1.

[0182] The results are as Figure 19 shown. The Y309 strain has strong inhibitory effects on the mycelial growth of the 12 tested plant pathogenic fungi. Among them, the inhibitory effects on Rhizoctonia cerealis, brown rot pathogen, and Ceratocystis fimbriata are the strongest, and the inhibition rates all exceed 70%, which are 80.9%, 76.1%, and 71.1% respectively. The inhibition rates on pathogenic fungi such as Colletotrichum destructivum, Fusarium verticillioides, Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, Fusarium pseudograminearum, and Botrytis cinerea are between 60.6% and 69.0%, which are 69.0%, 68.0%, 67.9%, 66.0%, 65.4%, 64.6%, 63.9%, and 60.6% respectively. The inhibition rate on Septoria apii is the lowest, only 50.2%. This indicates that the Y309 strain has strong broad-spectrum antibacterial properties.

[0183] (2) Antibacterial ability of strain Y309 against pathogenic bacteria

[0184] Use the double-layer plate method to determine the inhibitory effect of the Y309 strain on Pseudomonas syringae pv. lachrymans ( Pseudomonas syringae pv. Lachrymans ), Xanthomonas campestris pv. vesicatoria ( Xanthomonas campestris pv. Vesicatoria ), Ralstonia solanacearum ( Ralstonia solanacearum ), Xanthomonas citri subsp. citri ( Xanthomonas citri subsp. Citri ), Agrobacterium tumefaciens ( Agrobacterium vitis ), Pectobacterium carotovorum ( Pectobacterium carotovorum ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Salmonella ( SalmonellaThe growth inhibitory effects on 10 pathogenic bacteria such as Pseudomonas syringae pv. lachrymans (Smith) Young, Dye & Wilkie (sp.) were studied. A sterile toothpick was used to pick up the strain Y309 and inoculate it in the center of a PDA plate. After culturing at a constant temperature of 28 °C in an incubator for 5 days, 5 mL of chloroform was used to fumigate the strain upside down to kill it. After all the chloroform had volatilized, water agar containing a pathogenic bacteria solution with an OD 600 = 0.8 and 2% was poured onto the upper layer and placed in an incubator at 28 °C for constant temperature culture for 24 h. The diameter of the transparent antibacterial circle was measured by the cross method, and the diameter of the antibacterial circle was calculated according to formula (3).

[0185]

[0186] The results are as Figure 20 shown. The strain Y309 had the strongest inhibitory effects on Pseudomonas syringae pv. lachrymans and Bacillus cereus, and the diameters of the antibacterial circles exceeded 30 mm, being 34.75 mm and 32.17 mm respectively. The diameters of the antibacterial circles against Staphylococcus aureus, Salmonella spp., Xanthomonas campestris pv. vesicatoria (Doidge) Dye, Ralstonia solanacearum (Smith) Yabuuchi et al., and Xanthomonas citri subsp. citri (Hasse) Dowson were 21.71 mm, 21.44 mm, 20.58 mm, 13.51 mm, and 5.23 mm respectively. However, it had no antibacterial effect on Escherichia coli, Agrobacterium tumefaciens (Smith & Townsend) Conn, and Pectobacterium carotovorum subsp. carotovorum (Jones) Bergey et al.

[0187] Example 5 Growth promotion effect of strain Y309 on wheat

[0188] The wheat variety Jimai 22 (bred by Shandong Academy of Agricultural Sciences) was selected. The strain Y309 was inoculated into a liquid PD medium and cultured with shaking at 28 °C for 7 days to prepare a bacterial suspension of strain Y309. Wheat seeds were surface-sterilized by soaking in 75% alcohol for 30 s and rinsed 2 - 3 times with sterile water to wash away the residual alcohol. They were soaked in the original bacterial suspension and diluted solutions diluted 100-fold, 500-fold, and 1000-fold in the dark for 3 h, and then placed in a germination box for germination in the dark at 28 °C. 100 seeds were placed in each box to reduce errors, and each treatment was repeated 4 times. Sterile water treatment was used as a control. The germination potential and germination rate of the seeds were counted on the 3rd day and the 5th day respectively. The germination standard was that "the radicle was as long as or longer than the seed, and the plumule exceeded half of the seed". The bud length, root length, dry weight, and fresh weight of single-plant seedlings were counted on the 5th day. Seeds with obvious necrosis during this period should be picked out in time to avoid contamination.

[0189]

[0190] The results are as Figure 21As shown in the figure, the fermentation broth of strain Y309 with different dilution multiples has different degrees of influence on the growth of wheat. With the increase of the dilution multiple, the growth-promoting effect of the fermentation broth on the germination of wheat seeds and the growth of seedlings is significantly improved. The fermentation broth diluted 1000 times has the most significant growth-promoting effect on wheat seeds after soaking treatment. The germination potential, bud length, and root length of wheat in the fermentation broth stock solution treatment group are the lowest, which are reduced by 52.7%, 16.4%, and 27.9% respectively compared with the control treatment, indicating that the fermentation broth stock solution of strain Y309 has a significant inhibitory effect on the germination of wheat seeds and the growth of seedlings, and the concentration of plant growth substances produced is relatively high, which inhibits the growth of seeds. After soaking wheat seeds with the fermentation broth diluted 500 times for 3 days, the germination potential of wheat seeds can reach about 75%, which is significantly higher than that of other treatment groups. The germination potential of wheat in the fermentation broth treatment group diluted 1000 times is 75%, the bud length and root length can reach 95.07 mm and 91.25 mm, and the fresh weight reaches 0.19 g. Compared with the control group without inoculating the fermentation broth of the strain, all growth parameters have increased significantly. The germination potential, bud length, root length, and fresh weight of wheat are increased by 11.7%, 27.5%, 48.5%, and 26.7% respectively compared with the control. The treatment with the fermentation broth diluted 500 times has a certain growth-promoting effect on the germination potential and root length of wheat, while there is no significant difference in the bud length and fresh weight of wheat compared with the control group treatment.

[0191] Example 6 Determination of the plate control effect of strain Y309 on wheat sharp eyespot

[0192] The pathogen of the tested wheat sharp eyespot is Rhizoctonia cerealis Rhizoctonia solani ), and the wheat variety is the same as in Example 5. The preparation method of the Y309 strain bacterial suspension is the same as in Example 5. After surface disinfection of wheat seeds, soak them in sterile water for 3 h and then put them into a petri dish and keep them moist at 28 °C for germination for 48 h. Select wheat seeds with uniform size and consistent white tips and put them into a petri dish covered with Rhizoctonia cerealis. Put 10 seeds in each petri dish and pour a layer of sterile vermiculite on the petri dish. Use the root irrigation method to pour 100 μL of sterile water, the bacterial suspension stock solution, and the bacterial suspension dilutions diluted 5 times and 10 times into the roots of the seeds. Use 1000 times of 24% Jinggangmycin A aqueous agent as the positive control, put them in a sunlight greenhouse at 25 °C for cultivation, water regularly every day to keep the vermiculite moist, pull out the wheat roots after 14 days, and observe the degree of brown discoloration of the roots and leaf sheaths. The disease grading standard of wheat sharp eyespot is referred to the following statistics, and the disease index and control effect of each treatment are calculated using formulas 6) - 8).

[0193] Grade 0: No symptoms;

[0194] Grade 1: Leaf sheath turns brown;

[0195] Grade 2: There are obvious sharp eyespot lesions outside the leaf sheath, and the lesion diameter < 0.5 cm;

[0196] Level 3: There are one to several lesions outside the leaf sheath, and the diameter of the lesion > 0.5 cm;

[0197] Level 4: There are lesions inside the leaf sheath;

[0198] Level 5: There are lesions on the stem or the plant is dead.

[0199]

[0200] The results are as Figure 22 shown. The original fermentation broth of strain Y309 can effectively control wheat sharp eyespot. The control group of wheat inoculated only with Rhizoctonia cerealis showed severe disease, with obvious brown discoloration at the base of the stem. The incidence rate and disease index reached 80.0% and 36.00 respectively. The fermentation broth treatment group of the strain and the jinggangmycin treatment group could significantly reduce the incidence rate and disease index of wheat sharp eyespot, and the control effects were 52.8% and 50.0% respectively, with no significant difference between them. In contrast, the fermentation broth of strain Y309 still had a certain control effect on wheat sharp eyespot after being diluted 5 times. The incidence rate and disease index were reduced by 15.7% and 40.3% respectively compared with the control group treatment, and the relative control effect could reach 40.3%.

[0201] Example 7 Determination of the greenhouse control effect of strain Y309 against tomato gray mold

[0202] The pathogen of the tested tomato gray mold is Botrytis cinerea ( Botrytis cinerea ), and the tomato variety selected is Fenteli (Shouguang Gedun Agricultural Technology Co., Ltd.). The preparation method of the Y309 strain bacterial suspension is the same as that in Example 5.

[0203] Healthy and uniformly sized tomato leaves were picked from tomato plants grown to the six-leaf stage, washed 2 - 3 times with sterile water to remove surface impurities, and after natural drying, they were soaked in sterile water, the original bacterial suspension, and the bacterial suspension dilutions diluted 5 times and 10 times for 10 s respectively. After drying, 5 mm Botrytis cinerea fungal cakes were inverted and inoculated on the leaf surface. A 1000-fold dilution of 50% procymidone wettable powder was used as the positive control. The treated leaves were placed in a petri dish lined with 4 layers of moist filter paper and cultured in the dark at 22 °C for 3 d. Each treatment was set with 3 replicates, and each replicate had 10 leaves. After the control was completely diseased, photos were taken under bright field and ultraviolet light (365 nm) respectively. The area of the lesions was measured using Image J, and the control effect was calculated according to formula (9):

[0204]

[0205] The results are as Figure 23As shown in the figure, the fermentation broth of strain Y309 has a good control effect on tomato gray mold. The control effect of the original fermentation broth is slightly lower than that of procymidone, but the difference is not significant, both are above 65%. After the fermentation broth is diluted 5 times, it can still have a certain control effect on tomato gray mold, and the control effect is 27.9%.

[0206] Example 8 Determination of the greenhouse control effect of strain Y309 on pepper fusarium wilt

[0207] The pathogen of the tested pepper fusarium wilt is Fusarium oxysporum ( Fusarium oxysporum ), and the pepper variety selected is Zhongjiao No. 6 (bred by Beijing Academy of Agricultural Sciences). Preparation of inoculated soil: Inoculate Fusarium oxysporum into corn substrate, and culture it at 22 °C for about 10 days until the mycelium covers the whole medium. Then, mix the corn substrate full of the pathogen with the soil substrate (nutrient soil and vermiculite are mixed at a volume ratio of 1:1) evenly according to the addition amount of 3% (w / w%) to make the inoculated soil. The preparation method of the bacterial suspension of strain Y309 is the same as that in Example 5.

[0208] After the pepper seeds are disinfected, they are soaked in the original solution of the bacterial suspension of strain Y309, 500 times of 50% carbendazim wettable powder, and sterile water for 30 minutes respectively, and then sown. 4 seeds are sown in each pot, and there are 10 replicates for each treatment. The treatment with clear water without pathogenic bacteria is used as the control. 21 days after emergence, the peppers are pulled out by the roots, and the disease occurrence of the plants is observed. The disease grading standard of pepper fusarium wilt is statistically analyzed according to the severity of the discoloration of the vascular tissue as follows, and the disease index and control effect of each treatment are calculated.

[0209] Grade 0: The roots are healthy without symptoms;

[0210] Grade 1: The roots are slightly brown, and the brown area ≤ 33%;

[0211] Grade 2: The roots show rot, the brown area ≤ 67%, and there are lesions at the base of the stem;

[0212] Grade 3: The roots are severely rotten, the brown area ≥ 67%, and the plants wilt or even die.

[0213] The results are as Figure 24As shown, the fermentation broth of strain Y309 can effectively prevent and control the occurrence of pepper fusarium wilt. Compared with the healthy plants without pathogenic bacteria mixed in the soil, the incidence rate of the treatment group infected with Fusarium oxysporum reached 100%. The pepper plants were significantly dwarfed, and obvious root rot occurred. However, no obvious water shortage and chlorosis symptoms appeared in the above-ground leaves, and the disease index reached 38.73. Both the treatment groups of strain Y309 and carbendazim could significantly reduce the incidence rate of pepper fusarium wilt, and at the same time, the disease index decreased to 22.29 and 30.09. The control effect of strain Y309 could reach 40.0%, which was significantly higher than that of the carbendazim treatment group. The control effect of the carbendazim treatment group was only 22.3%. However, when measuring various growth indexes of pepper plants, although the plant height and root length of the fermentation broth treatment group were significantly higher than those of the sterile water and carbendazim treatment groups, they were lower than the control. The fresh weight per plant had no significant difference from that of the carbendazim treatment group and decreased by 38.0% compared with the CK treatment, indicating that strain Y309 could not completely relieve the growth inhibition effect of Fusarium oxysporum on tomatoes.

[0214] Example 9 Growth promotion effect of strain Y309 on corn in saline-alkali soil in Xinjiang

[0215] In this example, two groups of pot experiments were set up to further verify the growth promotion effect of strain Y309 on corn in saline-alkali soil in Xinjiang. The corn variety selected was Jinchong No. 1 (Xinjiang Meiyalianda Seed Industry Co., Ltd.). The preparation method of the strain Y309 bacterial suspension was the same as that in Example 5. The basic physical and chemical properties of saline-alkali soil and non-saline-alkali soil in Xinjiang are shown in Table 13. For each type of soil, 1.5 kg was used per pot, and the addition amount of the bacterial liquid was 50 mL. At the same time, in order to maintain the soil humidity, 100 mL of sterile water was added to the bacterial liquid. 2 g / kg of sodium chloride was added to the sterile water in the non-saline-alkali soil experiment. The sample soil was spread evenly on the kraft paper that was not easy to get wet, and then evenly sprayed. After evenly sprinkling the bacterial liquid on each layer on average, it was gently mixed, and soil disturbance was avoided as much as possible during this period until all the bacterial liquid was added. At the same time, the fermentation medium without inoculating the bacterial liquid was sprayed and mixed as the blank control. After the addition, the soil was weighed 1.5 kg and filled into plastic flower pots with a diameter of 18 cm. 3 corn seeds with consistent germination were sown in each pot, and the sowing depth was about 1 cm. After sowing, 100 mL of water was watered every two days. Thinning was started 3 days after emergence, and only two corn plants with uniform size were retained in each pot. 10 days after thinning, MS nutrient solution (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was supplemented once.

[0216] Table 13 Basic physical and chemical properties of different types of soil

[0217]

[0218] Samples were collected 40 days after sowing to detect the growth-promoting ability of the fermentation broth of strain Y309 on maize seedlings. The maximum leaf was measured three times from top to bottom using a handheld chlorophyll meter (SPAD-502PIU), and then the average SPAD value was used as the SPAD value of the maize plant. Growth indices such as maize seedling plant height, stem diameter, aboveground fresh weight, aboveground dry weight, maximum leaf area, and chlorophyll were measured.

[0219] The results are shown in Table 14. Strain Y309 had a good growth-promoting effect on the aboveground part of maize in Xinjiang saline-alkali soil ( Figure 25 ), which could increase the plant height of maize 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%.

[0220] Table 14 Growth-promoting effect of strain Y309 on maize in Xinjiang saline-alkali soil

[0221]

[0222] Note: Different lowercase letters indicate significant differences.

[0223] The results are shown in Table 15. Strain Y309 had a good growth-promoting effect on the aboveground and underground parts of maize in non-saline-alkali soil in Xinjiang with added salt ( Figure 26 ), which could increase the plant height of maize 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).

[0224] Table 15 Growth-promoting effect of strain Y309 on maize in non-saline-alkali soil in Xinjiang with added salt

[0225]

[0226] Note: Different lowercase letters indicate significant differences.

[0227] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

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

2. A microbial agent, biological pesticide or biological fertilizer prepared by the Streptomyces or its fermentation liquid according to claim 1.

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 preparing biofertilizer; 7) Used for plant growth promotion; 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 ); 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.).

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

6. Use of the Streptomyces according to claim 1 in resisting pathogens and plant diseases caused by pathogens: 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 include wheat sheath blight, tomato gray mold, and pepper wilt.

7. 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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