Streptomyces albidoflavus and application thereof

Abstract

The application provides a streptomyces albulus and application thereof, and relates to the technical field of microorganisms. Streptomyces alboflavus The streptomyces albulus is named as LY7, and the preservation number is CGMCC No. 37530. The strain has inhibiting effects on various plant pathogenic bacteria, and has great potential in agricultural applications such as plant disease control. The application expands the related application and product of the biocontrol bacteria streptomyces albulus, for example, a multifunctional microbial fertilizer taking mushroom dregs as a substrate, which has obvious bacteriostatic activity on the main soil-borne pathogenic bacteria of ginseng, can effectively prevent and control the occurrence of ginseng root diseases, can significantly promote the growth of ginseng and increase the yield of ginseng. In addition, the microbial fertilizer can effectively regulate the physical and chemical properties of ginseng land soil and improve the soil enzyme activity, and promote the healthy development of the ginseng industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Streptomyces chrysogenum and its applications. Background Technology

[0002] In ginseng cultivation, disease outbreaks can severely impact ginseng yield and quality; therefore, effective disease control is a crucial aspect of ginseng production. Diseases caused by Fusarium oxysporum (… F. oxysporum ) and robust red shell fungus ( I. robusta Soil-borne diseases of ginseng, such as root rot and rust rot, mainly damage the roots of ginseng. They can occur throughout the entire growth period of ginseng, leading to poor ginseng development, a sharp decline in yield, and seriously threatening the ginseng industry.

[0003] Traditionally, the control of ginseng diseases has relied primarily on chemical control. However, the long-term and continuous use of chemical pesticides has led to a series of serious consequences, such as the development of pesticide resistance in pathogens, reduced control effectiveness, environmental pollution, and threats to human health. Biological control, based on the use of beneficial microorganisms to control harmful organisms, avoids the series of plant protection, environmental, and energy problems associated with chemical pesticide use, avoids the harm of pesticide residues to humans and livestock, and promotes sustainable agricultural development.

[0004] Therefore, there is an urgent need in this field for biocontrol bacteria that can simultaneously target multiple ginseng diseases and effectively control multiple pathogens. By expanding the relevant applications of biocontrol bacteria, we can promote ginseng growth, increase ginseng yield, and promote the healthy development of the ginseng industry. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a strain of Streptomyces chrysogenum and its application, which can effectively prevent and control a variety of pathogens. By expanding the relevant applications of biocontrol bacteria, it can promote ginseng growth, increase ginseng yield, and promote the healthy development of the ginseng industry.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a strain of Streptomyces leucosus, the strain name of which is LY7, and it is classified as Streptomyces leucosus. Streptomyces alboflavus The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37530, deposited on January 26, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a fermentation broth comprising the aforementioned Streptomyces leucosus and a culture medium.

[0009] The present invention also provides a method for preparing the fermentation broth, comprising inoculating the Streptomyces leucosus into the culture medium and culturing it to obtain the fermentation broth.

[0010] The present invention also provides the application of the aforementioned Streptomyces flavus, the aforementioned fermentation broth, or the fermentation broth prepared by the aforementioned preparation method in any one or more of the following:

[0011] A1. Preparation of products for preventing and controlling ginseng diseases caused by pathogens.

[0012] A2. Prevention and control of ginseng diseases

[0013] A3. Promotes ginseng growth.

[0014] A4. Preparation of microbial fertilizer

[0015] A5. Regulate soil physical and chemical properties

[0016] A6. Preparation of soil conditioner.

[0017] Preferably, the pathogen includes Fusarium oxysporum. Fusarium oxysporum Fusarium solani Fusarium solani Strong Earth Red Shell Fungus Ilyonectria robusta Botrytis cinerea Botrytis cinerea Rhizoctonia solani Rhizoctonia solani ginseng phytotoxicum Phytophthora cactorum and Alternaria Alternaria alternata One or more of the following: the ginseng diseases include one or more of the following: ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot; the soil is the soil used for planting ginseng; the growth traits include ginseng yield and / or plant height.

[0018] The present invention also provides the application of the aforementioned Streptomyces flavus, the aforementioned fermentation broth, or the fermentation broth prepared by the aforementioned preparation method in any of the following:

[0019] B1. Control of one or more diseases including tobacco anthracnose, tobacco leaf spot, corn stalk rot, mango anthracnose, and raspberry leaf blight.

[0020] B2. Preparation of bacteria for the prevention and treatment of tobacco anthrax Colletotrichum destructivum Tobacco red spot bacterium Alternaria alternata Corn stalk rot fungus Fusarium verticillioides Mango anthracnose fungus Colletotrichum gloeosporioides and raspberry leaf blight fungus Coniothyrium fuckelii Any product containing any pathogenic bacteria.

[0021] This invention also provides a method for preparing microbial fertilizer, comprising the following steps:

[0022] The fermentation broth is prepared by mixing the Streptomyces chrysogenum, the fermentation broth, or the fermentation broth prepared by the preparation method with the enoki mushroom mycelium fermentation product.

[0023] The present invention also provides microbial fertilizer prepared by the aforementioned preparation method.

[0024] The present invention also provides the application of the microbial fertilizer obtained by the preparation method or the microbial fertilizer in any one or more of the following:

[0025] C1. Prevention and control of ginseng diseases

[0026] C2. Promotes ginseng growth.

[0027] C3. Repair the soil used for ginseng cultivation.

[0028] Preferably, the ginseng diseases include ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot; the growth traits include ginseng yield and / or plant height.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a strain of Streptomyces leucosus, named LY7, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 37530. This strain can effectively control a variety of plant pathogens and has great potential in agricultural applications such as plant disease control.

[0031] This invention, by expanding the applications of the biocontrol bacterium Streptomyces fulvicina, can significantly promote ginseng growth, increase ginseng yield, effectively regulate the physicochemical properties of ginseng-growing soil, and promote the healthy development of the ginseng industry.

[0032] The multifunctional microbial fertilizer based on *Flammulina velutipes* spawn substrate of this invention exhibits significant antibacterial activity against major soil-borne pathogens causing ginseng diseases, effectively preventing root diseases in ginseng. This microbial fertilizer also significantly promotes ginseng growth and increases yield. Furthermore, it effectively regulates the physicochemical properties of ginseng-growing soil and improves soil enzyme activity. Therefore, this microbial fertilizer based on *Flammulina velutipes* spawn substrate can be used for the biological control of ginseng root diseases, increasing yield without the drawbacks of chemical pesticides or the development of pesticide resistance in pathogens. It reduces the amount and frequency of chemical pesticide use and effectively utilizes the spawn substrate, solving the problem of spawn waste.

[0033] Biological Preservation Information:

[0034] The Streptomyces leucosus strain of this invention is named LY7 and is classified as Streptomyces leucosus. Streptomyces alboflavusThe strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37530, deposited on January 26, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0035] Figure 1 The images show the culture characteristics and scanning electron micrographs of Streptomyces leucopterus LY7 on Gao's No. 1 medium in Example 1. A shows the colony morphology of strain LY7, B shows the single colony morphology of strain LY7, C shows the electron micrograph of spore hyphae of strain LY7, and D shows the electron micrograph of spores of strain LY7.

[0036] Figure 2 The colony morphology of Streptomyces leucosus LY7 in Example 1 under different culture media and different culture days.

[0037] Figure 3 This is a multigene phylogenetic tree of Streptomyces LY7 from Example 1.

[0038] Figure 4 The image shows the inhibitory effect of Streptomyces leucosus LY7 on 12 pathogens in Example 2.

[0039] Figure 5 This is a response surface plot showing the effect of the interaction of nutritional factors on the antibacterial effect of Streptomyces leucovorum LY7 in Example 3.

[0040] Figure 6 This is a response surface plot showing the effect of the interaction of three environmental factors on the antibacterial effect of Streptomyces leucosus LY7 in Example 3.

[0041] Figure 7 This study evaluates the compatibility of the *Flammulina velutipes* mycelium residue extract with *Streptomyces leucosus* LY7 in Example 3.

[0042] Figure 8 This demonstrates the inhibitory effect of the bacterial fertilizer extract in Example 3 on the growth of major pathogens in ginseng roots.

[0043] Figure 9 The effect of microbial fertilizer on ginseng growth indicators in Example 3.

[0044] Figure 10 The microbial fertilizer in Example 3 demonstrates its efficacy in preventing root diseases in ginseng. Detailed Implementation

[0045] This invention provides a strain of Streptomyces leucosus, the strain name of which is LY7, and it is classified as Streptomyces leucosus. Streptomyces alboflavusThe strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37530, deposited on January 26, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] In this invention, the LY7 was isolated from farmland soil in Luolong District, Luoyang City, Henan Province. The LY7 of this invention grew well on Gao's No. 1 medium, with round colonies, neat edges, and tight adhesion to the medium; the aerial hyphae were orange-yellow in the center and white at the edges, with a fluffy, dense, and flat texture; the intramolecular hyphae were colorless, the colony surface was flat, and there was no pigment diffusion, consistent with the typical culture characteristics of Streptomyces.

[0047] The present invention also provides a fermentation broth comprising the LY7 and a culture medium. In the present invention, the culture medium comprises ISP2 medium; the preferred formulation of the ISP2 medium is the following components at the following mass-volume concentrations: 25.5 g / L sucrose, 19.3 g / L corn starch, 15.5 g / L yeast extract, and 1.6 g / L MgSO4·7H2O, wherein the solvent of the ISP2 medium is water.

[0048] The present invention also provides a method for preparing the fermentation broth, comprising inoculating the LY7 in the culture medium and culturing it to obtain the fermentation broth. In the preparation method of the fermentation broth of the present invention, the preferred culture conditions are as follows: pH is preferably 5.5-8, more preferably 6-7.5, more preferably 6.8-7.2, and most preferably 7.0; the culture medium bottling volume is preferably (50-100) mL / 250 mL, more preferably (60-80) mL / 250 mL, and more preferably 75 mL / 250 mL; the inoculum volume is preferably 1%-3%, more preferably 1%-2%, and more preferably 1%; the fermentation temperature is preferably 25℃-30℃, more preferably 26℃-29℃, more preferably 27℃ or 28℃, and most preferably 28℃; the fermentation speed is preferably 150 rpm-250 rpm, more preferably 180 rpm-220 rpm, and more preferably 200 rpm; the fermentation culture time is preferably 90 h-120 h, more preferably 95 h-115 h, more preferably 100 h-110 h, and most preferably 103 h. In a preferred embodiment, the fermentation broth is prepared as follows: The LY7 strain is inoculated at 1% into a shake flask with a culture medium volume of 75 mL / 250 mL, and fermented at pH 7.0, fermentation temperature 28℃, and rotation speed 200 rpm for 103 h. In this invention, the effective viable count in the prepared fermentation broth is greater than or equal to 2.95 × 10⁻⁶. 8 CFU·g -1 .

[0049] The present invention also provides the aforementioned Streptomyces chrysogenum. 、 The fermentation broth, or the fermentation broth prepared by the preparation method, may be used in any one or more of the following applications.

[0050] A1. Preparation of products for preventing and controlling ginseng diseases caused by pathogens.

[0051] A2. Prevention and control of ginseng diseases

[0052] A3. Promotes ginseng growth.

[0053] A4. Preparation of microbial fertilizer

[0054] A5. Regulate soil physical and chemical properties

[0055] A6. Preparation of soil conditioner.

[0056] Preferably, the pathogen includes Fusarium oxysporum. Fusarium oxysporum Fusarium solani Fusarium solani Strong Earth Red Shell Fungus Ilyonectria robusta Botrytis cinerea Botrytis cinerea Rhizoctonia solani Rhizoctonia solani ginseng phytotoxicum Phytophthora cactorum and Alternaria Alternaria alternata One or more of the following: the ginseng diseases include one or more of the following: ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot; the soil is the soil used for planting ginseng; the growth traits include ginseng yield and / or plant height.

[0057] The present invention also provides the aforementioned Streptomyces chrysogenum. 、 The fermentation broth or the fermentation broth prepared by the preparation method may be used in any of the following applications:

[0058] B1. Control of one or more diseases including tobacco anthracnose, tobacco leaf spot, corn stalk rot, mango anthracnose, and raspberry leaf blight.

[0059] B2. Preparation of drugs for the prevention and treatment of tobacco anthrax Colletotrichum destructivum Tobacco-borne disease Alternaria alternata Corn stalk rot Fusarium verticillioides Mango anthracnose Colletotrichum gloeosporioides and raspberry leaf blight Coniothyrium fuckelii Any product containing any pathogenic bacteria.

[0060] This invention uses the Streptomyces LY7 strain to... Fusarium oxysporum (Fusarium oxysporum, the pathogen causing ginseng root rot) 、Rhizoctonia solani(The pathogen causing ginseng damping-off - Rhizoctonia solani) Fusarium solani (Ginseng root rot pathogen - Fusarium solani) 、Phytophthora cactorum (Phytophthora infestans, the pathogen causing ginseng blight) 、 Alternaria alternata (Ginseng black spot fungus - Alternaria alternata) 、Fusarium verticillioides (Maize stalk rot fungus - Fusarium verticillatum) 、Colletotrichum destructivum (Tobacco anthrax bacteria - Destroy anthrax bacteria) 、 Coniothyrium fuckelii (Raspberry leaf blight pathogen - Vokk's shield) 、Alternaria alternata (Tobacco star causal agent - Alternaria) 、Ilyonectria robusta (Ginseng rust rot fungus - *Strongly vigorous* red shell fungus) 、Colletotrichum gloeosporioides (Mango anthracnose fungus) and Botytis cinerea All 12 pathogens (Gyromitra esculenta - Botrytis cinerea) showed significant inhibitory effects, with inhibition rates ranging from 60.13% to 91.82%. In addition to inhibiting ginseng-related pathogens, Streptomyces leucovorum also inhibited corn stalk rot pathogens. Fusarium verticillioides Tobacco anthrax bacteria Colletotrichum destructivum Tobacco red spot bacterium Alternaria alternata Mango anthracnose fungus Colletotrichum gloeosporioides and raspberry leaf blight fungus Coniothyrium fuckelii During growth, corresponding diseases and pests should be prevented and controlled.

[0061] This invention also provides a method for preparing microbial fertilizer, comprising the following steps: ... (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 、 The fermentation broth or the fermentation broth prepared by the above method is mixed with *Flammulina velutipes* mycelium fermentation substrate. The preferred mass-to-volume ratio of the *Flammulina velutipes* mycelium fermentation substrate to the *Streptomyces chrysogenum* fermentation broth is 0.5-3 g:1 mL, more preferably 1-2 g:1 mL, and even more preferably 1 g:1 mL. As an optional embodiment, the effective viable cell count in the fermentation broth is guaranteed to be greater than or equal to 2.95 × 10⁻⁶. 8 CFU·g -1 The preparation method of the microbial inoculant of this invention includes the following steps for preparing the *Flammulina velutipes* mushroom compost: adding urea, EM organic composting agent, and peanut bran to the *Flammulina velutipes* mushroom compost; manually turning and mixing the mixture evenly; adding water to adjust the moisture content of the mixture to 55%~60%; controlling the core temperature of the compost at 60~80℃; and fermenting the compost for 80~100 days. The preferred amount of urea added per cubic meter of *Flammulina velutipes* mushroom compost is 1~2 kg / m³. 3 Further preferred is 1.5 kg / m 3 The preferred addition amount of the EM organic matter composting agent is 100~200g / m³. 3 Further preferred is 100g / m 3The amount of peanut bran added is 100~200g / m³. 3 Further preferred is 100g / m 3 The composting fermentation time is further preferably 90 days.

[0062] This invention also provides a microbial fertilizer prepared by the aforementioned method. The effective viable count of the microbial fertilizer reaches 5.8 × 10⁻⁶. 9 CFU·g -1 .

[0063] The extract of the microbial fertilizer described in this invention showed a certain inhibitory effect on the mycelial growth of major pathogens causing diseases of ginseng roots at different dilution ratios. Specifically, it showed an inhibitory effect on... F. oxysporum The inhibition rate of mycelial growth was 79.28%–92.79%, and it had a significant effect on... R. solani The inhibition rate was 69.37%–99.10%, and for F. solani The inhibition rate was 50.52%–99.48%, and for I. robusta The inhibition rate was 70.31%–95.83%, and for B. cinerea The inhibition rate was 79.69%–99.48%. The best inhibition effect came from the original microbial fertilizer solution, which could inhibit the growth of pathogens causing major root diseases of ginseng by more than 90%.

[0064] The present invention also provides the application of the microbial fertilizer obtained according to the described microbial fertilizer or the preparation method in any one or more of the following:

[0065] C1. Prevention and control of ginseng diseases

[0066] C2. Promotes ginseng growth and increases ginseng yield.

[0067] C3. Repair the soil used for ginseng cultivation.

[0068] In this invention, the ginseng diseases are preferably ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot; the growth traits include ginseng yield and / or plant height.

[0069] In the application described in this invention, the microbial fertilizer treatment significantly reduces the occurrence of ginseng root rot, achieving a 62.55% control efficacy against ginseng root diseases. It also significantly promotes ginseng growth and has a marked yield-increasing effect, outperforming prebiotic fertilizer and enoki mushroom compost control. Furthermore, the microbial fertilizer effectively regulates the physicochemical properties of ginseng-growing soil and improves enzyme activity.

[0070] This invention does not specifically limit the product type. In the product, LY7, the fermentation broth, or the metabolites can all be the sole active ingredient, or other active ingredients may be included. The product of this invention can be used alone or in combination with other excipients and / or products. This invention does not specifically limit the concentration of the bacterial solution in specific applications; it can be conventionally selected according to actual needs.

[0071] The pathogens in the following embodiments of the present invention were all isolated, identified and preserved by the Green Prevention and Control Laboratory for Medicinal Plant Diseases of Jilin Agricultural University.

[0072] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0073] Example 1

[0074] Screening and identification of strain LY7

[0075] 1. Screening of strain LY7

[0076] A series of 321 actinomycetes were isolated and purified from 54 soil samples using a stepwise dilution method and Gao's No. 1 medium. *Fusarium ginseng* (Ginseng tip Fusarium) was among the isolates. F. oxysporum ) and Rhizoctonia solani ( R. solani Two main soil-borne pathogens of ginseng were used as target bacteria. The 321 biocontrol actinomycetes isolated and purified above were initially screened using the streak plate method. The selected strains showed resistance to... F. oxysporum Fourteen biocontrol actinomycetes were selected, all exhibiting inhibition rates exceeding 60%. Among them, strain LY7 (isolated from farmland soil in Luolong District, Luoyang City, Henan Province) showed the best inhibition rate. F. oxysporum The inhibitory effect was the best, with an average inhibition rate of 75.19%. Screening yielded [a specific inhibitor / treatment]. R. solani Fourteen strains showed an inhibition rate exceeding 80%, among which strain LY7 was the most effective. R. solani The average inhibition rate was 93.65%. Based on the comprehensive antibacterial ability test results, among the 321 actinomycete strains, [the following was observed]: ... R. solani and F. oxysporum Five strains showed an inhibition rate exceeding 60%. Among them, LY7 showed an inhibition rate of over 60%. F. oxysporum The inhibition rate reached 75.19%, and for R. solani The antibacterial rate reached 93.65%. Finally, strain LY7 was selected for subsequent experiments.

[0077] 2. Identification of strain LY7

[0078] Strawberry strain LY7 was streaked onto Gao's No. 1 medium, and its colony morphology was observed. Figure 1As shown: Strain LY7 grew well on Gao's No. 1 medium, with round colonies, neat edges, and tight adhesion to the medium; aerial hyphae were orange-yellow in the center and white at the edges, with a fluffy texture and dense, flat distribution; intramolecular hyphae were colorless, with a flat colony surface and no pigment diffusion; hyphae were approximately 0.6–0.9 μm wide, with some areas showing a network structure of interwoven hyphae; spore chains were attached to aerial hyphae, and the spores were short rod-shaped, approximately 0.5–0.7 μm × 0.9–1.2 μm in size, tightly arranged, with relatively long spore chains. Figure 1 ).

[0079] Strains of strain LY7 were streaked onto Czapek's agar, inorganic salt starch medium, yeast extract-malt extract medium, oat flake medium, potato extract medium, and nutrient agar, respectively, and cultured at 28°C for 7, 14, and 21 days, after which the culture characteristics were observed. Figure 2 ).

[0080] The morphological characteristics and physiological and biochemical properties of the strains were determined with reference to "Systematic Classification Techniques of Actinomycetes" (Chemical Industry Press, 2016) and "Rapid Identification and Systematic Classification of Actinomycetes" (Science Press, 2011). The identification results are shown in Table 1.

[0081] Table 1. Physiological and biochemical characteristics of strain LY7

[0082]

[0083] Note: A "+" reaction indicates a positive result, and a "-" reaction indicates a negative result.

[0084] This strain is a functional strain with a wide temperature range, moderate salt tolerance, and broad-spectrum acid and alkali adaptability. It can grow well at 4~45℃, pH 4~9 and 0~5% NaCl concentration, exhibiting outstanding environmental adaptability and stress resistance. The strain has a strong ability to utilize inorganic nitrogen, organic nitrogen and various soluble carbon sources, and can produce hydrolytic enzymes such as amylase and protease. It has the ability to decompose organic materials and convert nitrogen, and does not produce hydrogen sulfide, making it safe for application.

[0085] To achieve accurate classification and identification of strain LY7, its genomic DNA was used as a template, and PCR amplification was performed using actinomycete 16S rRNA gene-specific primers Act-235F (as shown in SEQ ID NO.1) and Act-878R (as shown in SEQ ID NO.2) to obtain a 16S rRNA gene fragment of approximately 640 bp (sequence shown in SEQ ID NO.3); simultaneously, primers were used... recA -730F (as shown in SEQ ID NO.4) and recA -1530R (as shown in SEQ ID NO.5) amplification obtained recAThe gene (approximately 850 bp, sequence as shown in SEQ ID NO. 6) was analyzed using primers. atpD -230F (as shown in SEQ ID NO.7) and atpD -680R amplification (as shown in SEQ ID NO. 8) atpD Gene (approximately 950 bp, sequence as shown in SEQ ID NO.9). This invention is based on 16S rRNA, recA and atpD Sequence alignment and multi-gene joint analysis of the three genes were performed to construct a multi-gene joint phylogenetic tree of strain LY7. Figure 3 ( ), in order to clarify its classification status.

[0086] The sequence of primer Act-235F is: 5'-CGCGGCCTATCAGCTTGTTG-3' (SEQ ID NO.1);

[0087] The sequence of primer Act-878R is: 5'-CCGTACTCCCCAGGCGGGG-3' (SEQ ID NO.2).

[0088] The 16S rRNA sequence of strain LY7 is as follows:

[0089] TCGCGGCCTTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGA(SEQ ID NO.3).

[0090] Primer recA The sequence of -730F is: 5'-GACATCGACAAGACCGAGAA-3' (SEQ ID NO.4);

[0091] Primer recA The sequence of -1530R is: 5'-TCGTCGATCTTGAAGTACGC-3' (SEQ ID NO.5).

[0092] For strain LY7 recA The sequence of the gene is as follows:

[0093] GGGGGCAGTCGCAGTGATGCGCATGGGCGAGCGGCCGAACGAGCCCATCGAGGTCATCCCCACCGGGTCGACCGCACTCGACGTCGCACTCGGCGTCGGCGGCATCCCCCGCGGCCGCGTGGTGGAGGTGTACGGCCCGGAGTCCTCCGGTAAGACGACCCTCACCCTGCACGCCGTGGCCAACGCCCAGCGGGCCGGTGGCGCGGTGGCCTTCGTGGACGCCGAGCACGCCCTCGACCCGGAGTACGCCAAGAAGCTCGGCGTGGACATCGACAACCTCATCCTGTCCCAGCCGGACAACGGCGAGCAGGCTCTCGAGATCGTCGACATGCTCGTCCGCTCCGGAGCCCTCGATCTGATCGTCATCGACTCCGTCGCCGCCCTGGTGCCGCGCGCGGAGATCGAGGGCGAGATGGGCGACTCGCACGTAGGTCTGCAGGCCCGCCTGATGAGCCAGGCGCTCCGGAAGATCACCAGCGCGCTCAACCAGTCCAAGACCACCGCGATCTTCATCAACCAGCTCCGCGAGAAGATCGGCGTGATGTTCGGCTCCCCGGAGACCACGACCGGTGGCCGCGCGCTGAAGTTCTACGCCTCCGTGCGCATGGACATCCGCCGCATCGAGACCCTCAAGGACGGCACGGACGCGGTGGGCAACCGCACCCGCGTCAAGGTCGTCAAGAACAAGGTCGCGCCCCCCTTCAAGCAGGCCGAGTTCGACATCCTCTACGGCCAGGGCATCAGCCGCGAGGGCGGCCTGATCGACATGGGCGTGGAGCACGGCTTCGTCCGCAAGGCCGGCGCTTGGTACACGTACGAGGGCGACCAGCTCGGCCAGTCAAGAGATCGCCC(SEQ ID NO.6).

[0094] Primer atpD The sequence of -230F is: 5'-GAYGAYCCNGARGTNATGAA-3' (SEQ ID NO.7);

[0095] Primer atpDThe sequence of -680R is: 5'-CCRTCNGCRTANGCCATCCA-3' (SEQ ID NO.8).

[0096] It is important to note here that primers atpD -230F and atpD -680R is for bacteria. atpD Degenerate primers designed for highly conserved gene regions, where Y represents C or T, R represents A or G, and N represents any one of A, T, C, and G.

[0097] strain LY7 atpD The gene sequence is as follows:

[0098] CTATGCTTGACAGCAATGTCTTCCTCGAGGTCATGTGGGCCGACCCGTCCACGGTCGCCGAGGCCGAGCGCTGGACGATCCACCGCAAGGCCCCGGCCTTCGACCAGCTCGAGTCCAAGACCGAGATGTTCGAGACCGGCCTGAAGGTCGTCGACCTTCTCACCCCGTACGTCAAGGGTGGAAAGATCGGTCTGTTCGGTGGTGCCGGTGTCGGCAAGACCGTGCTGATCCAGGAAATGATCGTCCGTGTGGCCAAGCTGCACGACGGTGTCTCCGTCTTCGCCGGTGTCGGCGAGCGCACCCGTGAGGGCAACGACCTCATGGTCGAGATGGAGGAAGCCGGCGTTCTGGACAAGACCGCGCTGGTCTTCGGCCAGATGGACGAGCCGCCGGGCACGCGTCTGCGCGTGGCCCTGGCCGGTCTGACCATGGCGGAGTACTTCCGCGATGTGCAGAAGCAGGACGTGCTGTTCTTCATCGACAACATCTTCCGCTTCACCCAGGCCGGTTCCGAGGTCTCGACCCTGCTCGGCCGCATGCCCTCCGCGGTGGGCTACCAGCCGAACCTGGCCGACGAGATGGGTCTCCTCCAGGAGCGCATCACCTCGACCCGTGGTCACTCGATCACCTCGATGCAGGCGATCTACGTCCCCGCGGACGACCTGACCGACCCGGCCCCGGCCACCACCTTCGCCCACCTCGACGCGACGACGGTGCTCTCCCGTCCGATCTCGGAGAAGGGCATCTACCCGGCCGTGGACCCGCTGGACTCCACGTCCCGCATCCTGGACCCGCGCTACATCGCGCAGGACCACTACGACGCCGCCATGCGCGTCAAGGGAATCCTGCAGAAGTACAAGGACCTCCAGGACATCATCGCGATCCTCGGCATCGACGAGCTCGGCGAAGAGGACAAGCTCGTCGTGTCCCGTGCAGCGTGCGTGTGGATGCGTCAG(SEQ ID NO.9).

[0099] Such as Figure 3 As shown, the LY7 strain and Streptomyces alboflavus MDJK44 and Streptomyces alboflavus NRRLB2373 clustered on the same branch, indicating a close phylogenetic relationship. The final identified strain, LY7, belongs to... Streptomyces alboflavus ( S. alboflavus ).

[0100] Based on morphological observation, physiological and biochemical index determination, and molecular sequencing and analysis results, LY7 was finally identified as Streptomyces flavus. Streptomyces alboflavus The strain LY7 was deposited on January 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37530, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0101] Example 2

[0102] To further clarify the activity of the strain, the antibacterial spectrum of Streptomyces leucovorum strain LY7, which showed significant inhibitory effects against two soil-borne pathogens of ginseng obtained from the initial screening, was determined.

[0103] The strain's resistance to [unclear] was determined using the plate two-well confrontation method. F. oxysporum, R. solani, Fusarium solani, Phytophthora cactorum, Alternaria alternata , Alternaria alternata , Fusarium verticillioides, Colletotrichum destructivum , Coniothyrium fuckelii , Alternaria alternata , Ilyonectria robusta , Colletotrichum gloeosporioides ,and Botrytis cinerea The inhibitory effects of a total of 12 pathogens were studied (all the target pathogens tested were isolated, identified and preserved by the Green Control Laboratory for Medicinal Plant Diseases of Jilin Agricultural University, and the information of the tested pathogens is shown in Table 2).

[0104] Table 2. Tested pathogenic fungi

[0105]

[0106] Studies have found that *Streptomyces flavus* LY7 exhibited broad-spectrum inhibitory effects against all 12 tested target pathogens. Figure 4 The inhibitory effects of Streptomyces leucovorum LY7 on 12 pathogens are shown in Table 3.

[0107] Table 3. Inhibitory effects of Streptomyces leucovorum LY7 on 12 pathogens.

[0108]

[0109] Depend on Figure 4It is evident that the *Streptomyces leucosus* strain LY7 exhibits broad-spectrum and significant antibacterial activity, showing varying degrees of inhibition against all 12 tested plant pathogens, with inhibition rates ranging from 60.13% to 91.82%. In summary, *Streptomyces leucosus* strain LY7 demonstrates strong inhibitory activity against multiple plant pathogenic fungi, exhibiting excellent overall antibacterial efficacy and promising potential for biocontrol applications.

[0110] Example 3

[0111] Creation and application of microbial fertilizers

[0112] 1. Optimization of shake-flask fermentation conditions for Streptomyces leucosus strain LY7

[0113] Single-factor experiments and response surface methodology were used to investigate the effects of these methods on the overall situation. F. oxysporum The antibacterial activity was used as the response value to optimize the shake-flask fermentation conditions of Streptomyces leucosus strain LY7.

[0114] First, ISP2 was selected as the basic culture medium from 10 fermentation media (Table 4) (culture medium formula: 4 g yeast powder, 10 g malt extract, 4 g glucose and 1000 mL distilled water).

[0115] Table 4. Formulas of the basal culture medium used in the test

[0116]

[0117] The composition of the culture medium was further optimized on ISP2 basal medium. Single-factor screening experiments were conducted on the types and concentrations of carbon sources, nitrogen sources, and inorganic salts.

[0118] The types of carbon sources selected are: sucrose, corn flour, soluble starch, glucose, maltose, and glycerol; the concentrations of the carbon sources selected are: 0%, 1%, 1.5%, 2%, 2.5%, and 3%.

[0119] The types of nitrogen and carbon sources selected are: yeast powder, beef extract, peptone, soybean flour, potassium nitrate, and urea; the concentrations of nitrogen and carbon sources selected are: 0%, 1%, 1.5%, 2%, 2.5%, and 3%.

[0120] The types of inorganic salts selected are: ZnSO4·7H2O, KH2PO4, MgSO4·7H2O, FeSO4, CaCl2 and MnSO4·H2O; the concentrations of the inorganic salts selected are: 0%, 0.05%, 0.1%, 0.15%, 0.2% and 0.25%.

[0121] Based on the single-factor experiments, the optimal fermentation medium formulation was further determined through response surface methodology: 25.5 g / L sucrose, 19.3 g / L corn starch, 15.5 g / L yeast extract, and 1.6 g / L MgSO4·7H2O, with deionized water as the solvent.

[0122] On the other hand, based on the determination of the optimal fermentation medium formulation, single-factor and response surface methodology experiments were also conducted on the environmental conditions (inoculum size, culture temperature, initial pH, bottle volume, shaker speed, and culture time) for shake flask culture. Ultimately, the optimal shake flask culture conditions for *Streptomyces leucosus* strain LY7 were determined to be: pH 7.0, bottle volume 75 mL / 250 mL, 1% inoculum size, 28℃, and culture at 200 rpm for 103 h. Figure 5 , Figure 6 ).

[0123] 2. Creation and application of microbial fertilizers

[0124] (1) Composting and fermentation of enoki mushroom substrate

[0125] The fermentation of mushroom residue was carried out in accordance with the Technical Specification for Fermentation of Edible Fungi Residue (NY / T 3291-2018).

[0126] Add 1.5 kg of urea, 100 g of EM organic composting agent (purchased from Shandong Fengbole Agricultural Co., Ltd.; registration certificate number: Microbial Fertilizer (2021) No. 10618), and 100 g of peanut bran to each cubic meter of enoki mushroom substrate (provided by Changchun Xuerong Biotechnology Co., Ltd.). Add water to adjust the moisture content of the mixture to 55%~60%, and mix thoroughly by hand. Then stack the mixture into strip-shaped fermentation piles with a base width of about 2.5~3 m, a top width of 0.5~1 m, a height of 1~1.2 m, and a length of 7~8 m, and cover the surface with a layer of plastic film. Before turning the pile each day, use a probe thermometer to randomly measure the temperature at five points about 40~50 cm high. Turn the pile every 3~4 days to ensure oxygen supply. Utilize the heat generated by aerobic fermentation of microorganisms to allow the pile to naturally warm up and maintain a temperature range of 60℃~80℃. When the pile temperature equals the ambient temperature, composting is complete. The mixture enters a high-temperature period when the temperature rises to 60℃ on the 3rd day of microbial fertilizer production, and enters a cooling period after reaching a maximum temperature of 80℃ on the 26th day. It then enters the late stage of decomposition after dropping to 30℃ on the 80th day. The mixture is turned over 20 times in total, taking 90 days.

[0127] (2) Compatibility evaluation of Streptomyces leucanthin LY7 and enoki mushroom substrate

[0128] Preparation of Flammulina velutipes mycelium residue extract: Flammulina velutipes mycelium fermentation material was dehydrated and dried to constant weight in an 80 ℃ oven, then pulverized to obtain Flammulina velutipes mycelium residue powder, which was set aside. 5 g of Flammulina velutipes mycelium residue powder was weighed and added to 45 mL of sterile water for dark extraction for 48 h. The extract was then centrifuged to remove the precipitate, and filtered through a sterile filter membrane to obtain the Flammulina velutipes mycelium residue extract.

[0129] The compatibility test between the obtained Streptomyces leucovorum LY7 and the extract of Flammulina velutipes was conducted using the inhibition zone method. Streptomyces leucovorum LY7 was cultured on ISP2 (International Streptomyces Project) medium in a shaker for 72 h. The bacterial suspension was then spread onto PDA medium. Filter paper discs were placed on the culture dishes coated with the bacterial suspension. 5 μL of the stock extract, 10-fold and 20-fold dilutions of the extract were respectively added to the filter paper discs, with each treatment repeated three times. An equal volume of sterile water was added as a blank control.

[0130] After testing, it passed. Figure 7 It is known that the extract of enoki mushroom substrate has no inhibitory effect on the strain, and Streptomyces leucovorum LY7 can be used to create microbial fertilizers.

[0131] (3) Creation of microbial fertilizers

[0132] The enoki mushroom mycelium fermentation substrate was mixed with Streptomyces chrysogenum LY7 fermentation broth (2.95 × 10⁻⁶) at ratios of 3:1, 2:1, and 1:1 (w:v = g:mL). 8 CFU·g -1 They are mixed together to obtain microbial fertilizer.

[0133] The created microbial fertilizer was thoroughly mixed with sterile water at a ratio of 1:9 (g / mL) and allowed to stand in the dark for 48 h. The precipitate was then removed by centrifugation, and the extract was filtered through sterile gauze to obtain the fertilizer extract. Its activity against *Fusarium oxysporum* (ginseng) was determined using the plate confrontation method. F. oxysporum The antibacterial activity of the compound was investigated to screen for the optimal ratio.

[0134] The optimal ratio of *Flammulina velutipes* mycelium fermentation substrate to *Streptomyces leucanthin* LY7 fermentation broth was determined to be 1:1 (w:v = g:mL). At this ratio, the effective viable count of the microbial fertilizer reached 5.8 × 10⁻⁶. 9 CFU·g -1 For ginseng Fusarium oxysporum ( F. oxysporum The antibacterial rate reached 77.14%.

[0135] The extracts of the microbial fertilizer were diluted 10, 100, and 1000 times for later use. PDA medium cooled to 40 °C was mixed with the above-mentioned microbial fertilizer extracts of different concentrations at a ratio of 9:1 (v / v), thoroughly shaken, and poured into sterile petri dishes to prepare microbial plates. A mycelial cake of the main ginseng pathogenic fungus with a diameter of 8.0 mm was inoculated in the center of each microbial plate. PDA plates without microbial fertilizer extract served as controls. Each treatment was repeated three times. The colony diameter was measured using the cross-cross method to determine the growth inhibition rate of different concentrations of microbial fertilizer extracts against the main root pathogenic fungi of ginseng.

[0136] Table 4 and Figure 8 The results showed that the bacterial fertilizer extract exhibited a certain inhibitory effect on the mycelial growth of the main pathogens causing diseases of ginseng roots at different dilution ratios.

[0137] Among them F. oxysporum The inhibition rate of mycelial growth was 79.28%–92.79%, and it had a significant effect on... R. solani The inhibition rate was 69.37%–99.10%, and for F. solani The inhibition rate was 50.52%–99.48%, and for I. robusta The inhibition rate was 70.31%–95.83%, and for B. cinerea The inhibition rate was 79.69%–99.48%.

[0138] Microbial fertilizer extract using enoki mushroom substrate as a base is effective against Fusarium oxysporum and Fusarium solani, which cause root rot in ginseng. (F. oxysporum) , F. solani ), the robust red-shelled fungus that causes ginseng rust disease ( I. robusta Botrytis cinerea, which causes gray mold in ginseng ( B. cinerea ) and Rhizoctonia solani, which causes ginseng damping-off disease ( R. solani All of them exhibited significant antibacterial activity (Table 5, Figure 8 ).

[0139] Table 5. Inhibitory effects of different dilutions of bacterial fertilizer extract on the growth of pathogens causing major root diseases in ginseng.

[0140]

[0141] Note: Different lowercase letters after the data in the table indicate that the results were obtained using the Tukyr T (HSD) test. P <0.05 level difference significance.

[0142] (3) Evaluation of the field application effect of microbial fertilizer

[0143] The experiment was conducted in Fusong, and a total of 6 treatments were set up, including 1 blank control group (CK) and 5 microbial fertilizer treatment groups.

[0144] The specific treatment plan is as follows: A control group (CK) was set up in the experiment, using 400g / m³ of microbial fertilizer. 2 Microbial fertilizer 600g / m 2 Microbial fertilizer 800g / m 2 Probiotic fertilizer (manufacturer: Shandong Jingqing Agriculture, product number: JQ-YSY-GB-40kg) 300g / m³ 2 Enoki mushroom substrate 600g / m 2 There were 6 treatments, each with 3 replicates, for a total of 18 cells, each cell area being 3m². 2 The plots are randomly arranged. Before sowing, the microbial fertilizer is evenly spread on the ground surface, and then the fertilizer is mixed into the topsoil by mechanical tillage.

[0145] The results showed that microbial inoculant treatment significantly promoted ginseng growth and had a marked yield-increasing effect, which was superior to prebiotic fertilizer and enoki mushroom substrate control, especially at 800 g / m³. 2 Under this treatment, ginseng plant height, stem length, stem diameter, and other growth indicators showed outstanding performance, and the yield increase was significantly higher than other treatments, demonstrating the best overall effect on ginseng growth and yield (as shown in Tables 6 and 7). Figure 9 ).

[0146] Table 6. Effects of microbial inoculants on the aboveground growth indicators of ginseng.

[0147]

[0148] Note: Different lowercase letters after the data in the table indicate that the results were obtained using the Tukyr T (HSD) test. P <0.05 level difference significance.

[0149] Table 7. Effects of microbial inoculants on ginseng root growth indicators and yield.

[0150]

[0151] Note: Different lowercase letters after the data in the table indicate that the results were obtained using the Tukyr T (HSD) test. P <0.05 level difference significance.

[0152] Meanwhile, microbial fertilizer can significantly reduce the occurrence of ginseng root rot at 800 g / m³. 2 The treatment showed the highest average control efficacy, achieving 62.55% control over ginseng root diseases (Table 8). Figure 10 In addition, microbial fertilizers can effectively regulate the physicochemical properties of ginseng-growing soil and improve enzyme activity, at 800 g / m³. 2 The performance of each indicator under the treatment is outstanding (as shown in Tables 9 and 10).

[0153] Table 8. The preventive effect of microbial fertilizer on ginseng root diseases.

[0154]

[0155] Note: Different lowercase letters after the data in the table indicate that the differences are significant at the P < 0.05 level according to the Tuky T (HSD) test.

[0156] Table 9. Effects of microbial inoculants on the physicochemical properties of ginseng rhizosphere soil.

[0157]

[0158] Note: Different lowercase letters after the data in the table indicate that the results were obtained using the Tukyr T (HSD) test. P <0.05 level difference significance.

[0159] Table 10 Effects of microbial inoculants on enzyme activity in ginseng rhizosphere soil

[0160]

[0161] Note: Different lowercase letters after the data in the table indicate that the results were obtained using the Tukyr T (HSD) test. P <0.05 level difference significance.

[0162] The results of the above embodiments indicate that the microbial fertilizer prepared by combining *Streptomyces leucovorum* strain LY7 with *Flammulina velutipes* spawn can target *Fusarium oxysporum* and *Fusarium solani*, which cause root rot in ginseng. (F. oxysporum) , F. solani ), the robust red-shelled fungus that causes ginseng rust disease ( I. robusta Botrytis cinerea, which causes gray mold in ginseng ( B. cinerea ) and Rhizoctonia solani, which causes ginseng damping-off disease ( R. solani All of them have obvious antibacterial activity; they significantly promote ginseng growth and have a significant yield-increasing effect, with growth-promoting and yield-increasing effects that are significantly better than those of probiotic fertilizer and enoki mushroom substrate; in addition, this microbial fertilizer significantly reduces the occurrence of ginseng root rot and can also effectively regulate the physicochemical properties of ginseng soil and improve soil enzyme activity.

[0163] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Streptomyces chrysophagus ( Streptomyces alboflavus LY7, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37530, deposited on January 26, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A fermentation broth, characterized in that, It comprises the Streptomyces leucosus of claim 1 and a culture medium.

3. The method for preparing fermentation broth as described in claim 2, characterized in that, This includes inoculating the Streptomyces leucosus into the culture medium and culturing it to obtain a fermentation broth.

4. The use of the *Streptomyces chrysogenum* as described in claim 1 or the fermentation broth as described in claim 2 in any one or more of the following: A1. Preparation of products for preventing and controlling ginseng diseases caused by pathogens. A2. Prevention and control of ginseng diseases A3. Promotes ginseng growth. A4. Preparation of microbial fertilizer A5. Regulate soil physical and chemical properties A6. Preparation of soil conditioner; The pathogen is Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum Fusarium solani ( ), Fusarium solani ), and robust red shell fungus ( Ilyonectria robusta ), Botrytis cinerea ( Botrytis cinerea ), Rhizoctonia solani ( Rhizoctonia solani ), Phytophthora ( Phytophthora cactorum ) and Alternaria ( Alternaria alternata One or more of the following; The ginseng diseases mentioned are one or more of the following: ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot. The soil in question is soil used for growing ginseng; The growth traits mentioned are ginseng yield and / or plant height.

5. The use of the Streptomyces chrysogenum as described in claim 1 or the fermentation broth as described in claim 2 in any of the following: B1. Control of one or more of the following diseases: tobacco anthracnose, tobacco leaf spot, corn stalk rot, mango anthracnose, and raspberry leaf blight. B2. Preparation of bacteria for the prevention and control of tobacco anthrax ( Colletotrichum destructivum ), tobacco red spot bacterium ( Alternaria alternata ), corn stalk rot fungus ( Fusarium verticillioides Mango anthracnose fungus ( Colletotrichum gloeosporioides ) and raspberry leaf blight fungus ( Coniothyrium fuckelii Products containing any of the pathogens listed in the list.

6. A method for preparing a microbial fertilizer, characterized in that, Includes the following steps: The mixture is prepared by mixing the Streptomyces cylindrica described in claim 1 or the fermentation broth described in claim 2 with the enoki mushroom mycelium fermentation product.

7. The microbial fertilizer prepared by the method according to claim 6.

8. The use of the microbial fertilizer according to claim 7 in any one or more of the following: C1. Prevention and control of ginseng diseases C2. Promotes ginseng growth. C3. Repair the soil used for ginseng cultivation; The ginseng diseases mentioned are one or more of the following: ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot. The growth traits mentioned are ginseng yield and / or plant height.

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