Streptomyces albidoflavus and application thereof

Abstract

The invention provides streptomyces albidoflavus and application thereof, and relates to the technical field of microorganisms. The streptomyces alboflavus disclosed by the invention is named as Streptomyces alboflavus LY7, and the preservation number of the streptomyces alboflavus LY7 is CGMCC (China General Microbiological Culture Collection Center) No.37530. The strain has an inhibition effect on various phytopathogens, and has great potential in agricultural applications such as plant disease control. Related application and products of the biocontrol bacterium streptomyces albidoflavus, such as multifunctional bacterial manure taking needle mushroom bran as a matrix, are expanded, and the streptomyces albidoflavus has obvious bacteriostatic activity on main soil-borne disease pathogenic bacteria of ginseng, and can effectively prevent and treat root diseases of ginseng; the ginseng growth can be obviously promoted; the ginseng yield is increased. In addition, the microbial fertilizer can also effectively regulate the physicochemical properties of soil in the ginseng field, improve the enzyme activity of the soil and promote the healthy development of the ginseng industry.

Description

A strain of Streptomyces chrysogenum and its application 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, diseases can severely impact yield and quality, making effective disease control a crucial aspect of ginseng production. Soil-borne diseases of ginseng, such as root rot and rust rot caused by *Fusarium oxysporum* and *I. robusta*, primarily affect the roots and can occur throughout the entire growth cycle, leading to poor ginseng development, a sharp decline in yield, and a serious threat to 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: The present invention provides a Streptomyces alboflavus strain, the strain name of which is LY7, classified as Streptomyces alboflavus, deposited at the China General Microbiological Culture Collection Center, with the strain accession number CGMCC No. 37530, the deposit date being January 26, 2026, and the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a fermentation broth comprising the aforementioned Streptomyces leucosus and a culture medium.

[0008] 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.

[0009] The present invention also provides the application of the Streptomyces cylindrica or the fermentation broth or the fermentation broth prepared by the preparation method in any one or more of the following: A1, preparing products for preventing and controlling ginseng diseases; A2, preventing and controlling ginseng diseases; A3, promoting ginseng growth; A4, preparing microbial fertilizer; A5, regulating soil physicochemical properties; A6, preparing soil conditioner.

[0010] Preferably, the pathogenic fungi include one or more of the following: *Fusarium oxysporum*, *Fusarium solani*, *Ilyonectria robusta*, *Botrytis cinerea*, *Rhizoctonia solani*, *Phytophthora cactorum*, and *Alternaria alternata*; 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 soil used for planting ginseng; and the growth traits include ginseng yield and / or plant height.

[0011] This invention also provides the application of the aforementioned Streptomyces leucanthin or the fermentation broth or the fermentation broth prepared by the aforementioned preparation method in any of the following: B1, controlling one or more diseases of tobacco anthracnose, tobacco red spot disease, corn stalk rot, mango anthracnose and raspberry leaf blight; B2, preparing a product for controlling any one of the pathogens of tobacco anthracnose fungus Colletotrichum destructivum, tobacco red spot fungus Alternaria alternata, corn stalk rot fungus Fusarium verticillioides, mango anthracnose fungus Colletotrichum gloeosporioides and raspberry leaf blight fungus Coniothyrium fuckelii.

[0012] The present invention also provides a method for preparing microbial fertilizer, comprising the following steps: mixing the Streptomyces chrysogenum or the fermentation broth or the fermentation broth prepared by the preparation method with the enoki mushroom mycelium fermentation product.

[0013] The present invention also provides microbial fertilizer prepared by the aforementioned preparation method.

[0014] This 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: C1, prevention and control of ginseng diseases; C2, promotion of ginseng growth; C3, restoration of soil for ginseng cultivation.

[0015] 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.

[0016] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a strain of Streptomyces leucosus, named LY7, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with 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.

[0017] 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.

[0018] 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.

[0019] Biological Preservation Information: The Streptomyces alboflavus strain of this invention, with the strain name LY7 and classification name Streptomyces alboflavus, is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 37530, on January 26, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0020] Figure 1 shows the culture characteristics and scanning electron micrographs of Streptomyces leucosus LY7 on Gao's No. 1 medium in Example 1. In the figure, A is a colony morphology image of strain LY7, B is a single colony morphology image of strain LY7, C is an electron micrograph of spore hyphae of strain LY7, and D is an electron micrograph of spores of strain LY7.

[0021] Figure 2 shows the colony morphology of Streptomyces whitworthii LY7 ​​in Example 1 under different culture media and different culture days.

[0022] Figure 3 is a multigene phylogenetic tree of Streptomyces LY7 in Example 1.

[0023] Figure 4 shows the inhibitory effect of Streptomyces leucovorum LY7 on 12 pathogens in Example 2.

[0024] Figure 5 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.

[0025] Figure 6 is a response surface plot showing the effect of the interaction of three environmental factors on the antibacterial effect of Streptomyces leucovorum LY7 in Example 3.

[0026] Figure 7 shows the compatibility evaluation of the *Flammulina velutipes* mycelium residue extract and *Streptomyces leucosus* LY7 in Example 3.

[0027] Figure 8 shows the inhibitory effect of the bacterial fertilizer extract in Example 3 on the growth of major pathogens in ginseng roots.

[0028] Figure 9 shows the effect of microbial fertilizer on ginseng growth indicators in Example 3.

[0029] Figure 10 shows the preventive effect of microbial fertilizer on ginseng root diseases in Example 3. Detailed Implementation

[0030] This invention provides a Streptomyces alboflavus strain, named LY7, classified as Streptomyces alboflavus, 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.

[0031] 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.

[0032] 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.

[0033] 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 .

[0034] The present invention also provides the application of the aforementioned Streptomyces chrysogenum, the fermentation broth, and the fermentation broth prepared by the preparation method in any one or more of the following: A1, preparing products for preventing and controlling ginseng diseases; A2, preventing and controlling ginseng diseases; A3, promoting ginseng growth; A4, preparing microbial fertilizer; A5, regulating soil physicochemical properties; A6, preparing soil conditioner.

[0035] Preferably, the pathogenic fungi include one or more of the following: *Fusarium oxysporum*, *Fusarium solani*, *Ilyonectria robusta*, *Botrytis cinerea*, *Rhizoctonia solani*, *Phytophthora cactorum*, and *Alternaria alternata*; 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 soil used for planting ginseng; and the growth traits include ginseng yield and / or plant height.

[0036] This invention also provides the application of the aforementioned *Streptomyces leucopsis*, the fermentation broth, or the fermentation broth prepared by the aforementioned method in any of the following: B1, controlling one or more diseases of tobacco anthracnose, tobacco red spot disease, corn stalk rot, mango anthracnose, and raspberry leaf blight; B2, preparing a product for controlling any one of the pathogens of tobacco anthracnose (*Colletotrichum destructivum*), tobacco red spot disease (*Alternaria alternata*), corn stalk rot disease (*Fusarium verticillioides*), mango anthracnose (*Colletotrichum gloeosporioides*), and raspberry leaf blight disease (*Coniothyrium fuckelii*).

[0037] This invention utilizes the aforementioned *Streptomyces LY7* strain to combat *Fusarium oxysporum* (the pathogen causing ginseng root rot), *Rhizoctonia solani* (the pathogen causing ginseng damping-off), *Fusarium solani* (the pathogen causing ginseng root rot), *Phytophthora cactorum* (the pathogen causing ginseng blight), *Alternaria alternata* (the pathogen causing ginseng black spot), *Fusarium verticillioides* (the pathogen causing corn stalk rot), *Colletotrichum destructivum* (the pathogen causing tobacco anthracnose), *Coniothyrium fuckelii* (the pathogen causing raspberry leaf blight), *Alternaria alternata* (the pathogen causing tobacco red spot), and *Ilyonectria*. The *Streptomyces robusta* (the causal agent of ginseng rust rot), *Colletotrichum gloeosporioides* (the causal agent of mango anthracnose), and *Botytis cinerea* (the causal agent of ginseng gray mold) all showed significant inhibitory effects, with inhibition rates ranging from 60.13% to 91.82%. In addition to inhibiting ginseng-related pathogens, *Streptomyces gloeosporioides* also inhibited the growth of *Fusarium verticillioides* (the causal agent of corn stem rot), *Colletotrichum destructivum* (the causal agent of tobacco anthracnose), *Alternaria alternata* (the causal agent of tobacco red spot), *Colletotrichum gloeosporioides* (the causal agent of mango anthracnose), and *Coniothyrium fuckelii* (the causal agent of raspberry leaf blight), thus enabling the control of these diseases.

[0038] This invention also provides a method for preparing microbial fertilizer, comprising the following steps: mixing the *Streptomyces chrysogenum*, the fermentation broth, or the fermentation broth prepared by the method 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 count in the fermentation broth is guaranteed to be greater than or equal to 2.95 × 10⁻⁶. 8 CFU·g -1The 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 3 The 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.

[0039] 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 .

[0040] The extract of the microbial fertilizer described in this invention exhibited a certain inhibitory effect on the mycelial growth of major pathogens causing diseases of ginseng roots at different dilution ratios. Specifically, the inhibition rates were 79.28–92.79% against *F. oxysporum*, 69.37–99.10% against *R. solani*, 50.52–99.48% against *F. solani*, 70.31–95.83% against *I. robusta*, and 79.69–99.48% against *B. cinerea*. The best inhibitory effect was observed in the undiluted microbial fertilizer, achieving an inhibition rate of over 90% against the growth of major root pathogens causing diseases of ginseng.

[0041] This invention also provides the application of the microbial fertilizer obtained according to the microbial fertilizer or the preparation method described herein in any one or more of the following: C1, prevention and control of ginseng diseases; C2, promotion of ginseng growth and increase of ginseng yield; C3, restoration of soil for ginseng cultivation.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1: Screening and Identification of Strains LY7 1. Screening of Strains LY7: A serial dilution isolation method was used with Gao's No. 1 medium to isolate and purify 321 actinomycetes from 54 soil samples. Using *Fusarium oxysporum* and *R. solani*, two major soil-borne pathogens of ginseng, as target bacteria, the 321 biocontrol actinomycetes were initially screened using the streak plate method. Fourteen biocontrol actinomycetes showed inhibition rates of over 60% against *F. oxysporum*. Among them, strain LY7 (isolated from farmland soil in Luolong District, Luoyang City, Henan Province) showed the best inhibitory effect on *F. oxysporum*, with an average inhibition rate of 75.19%. Fourteen strains showed inhibition rates of over 80% against *R. solani*, with strain LY7 showing an average inhibition rate of 93.65% against *R. solani*. Based on the comprehensive antibacterial ability test results, among the 321 actinomycete strains, 5 strains showed inhibition rates exceeding 60% against both *R. solani* and *F. oxysporum*. Among them, LY7 showed an inhibition rate of 75.19% against *F. oxysporum* and 93.65% against *R. solani*. Strain LY7 was ultimately selected for subsequent experiments.

[0048] 2. Identification of strain LY7: Strain LY7 was streaked onto Gao's No. 1 medium, and its colony morphology was observed, as shown in Figure 1. Strain LY7 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 middle and white at the edges, with a fluffy texture and dense, flat distribution. The intramolecular hyphae were colorless, and the colony surface was flat with no pigment diffusion. The hyphal width was approximately 0.6~0.9μm, and in some areas, the hyphae were interwoven into a network structure. The spore chains were attached to the aerial hyphae, and the spores were short rod-shaped, approximately 0.5~0.7μm × 0.9~1.2μm in size, tightly arranged, and the spore chains were relatively long (Figure 1).

[0049] 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, and their culture characteristics were observed (Figure 2).

[0050] 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.

[0051] Table 1. Physiological and biochemical characteristics of strain LY7

[0052] Note: A "+" reaction indicates a positive result, and a "-" reaction indicates a negative result.

[0053] 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.

[0054] To achieve accurate classification and identification of strain LY7, its genomic DNA was used as a template for PCR amplification using the 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), yielding a 16S rRNA gene fragment of approximately 640 bp (sequence shown in SEQ ID NO.3). Simultaneously, the recA gene (approximately 850 bp, sequence shown in SEQ ID NO.6) was amplified using primers recA-730F (as shown in SEQ ID NO.4) and recA-1530R (as shown in SEQ ID NO.5), and the atpD gene (approximately 950 bp, sequence shown in SEQ ID NO.9) was amplified using primers atpD-230F (as shown in SEQ ID NO.7) and atpD-680R (as shown in SEQ ID NO.8). This invention uses sequence alignment and multi-gene joint analysis of three genes, 16S rRNA, recA, and atpD, to construct a multi-gene joint phylogenetic tree of strain LY7 (Figure 3) to clarify its taxonomic position.

[0055] The sequence of primer Act-235F is: 5'-CGCGGCCTATCAGCTTGTTG-3' (SEQ ID NO.1); the sequence of primer Act-878R is: 5'-CCGTACTCCCCAGGCGGGG-3' (SEQ ID NO.2).

[0056] The sequence of the 16S rRNA of strain LY7 is as follows: TCGCGGCCTTATCAGCTTGTTGGTGAGGTAGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGA (SEQ ID NO.3).

[0057] The sequence of primer recA-730F is: 5'-GACATCGACAAGACCGAGAA-3' (SEQ ID NO.4); the sequence of primer recA-1530R is: 5'-TCGTCGATCTTGAAGTACGC-3' (SEQ ID NO.5).

[0058] The sequence of the recA gene of strain LY7 is as follows: GGGGGGCAGTCGCAGTGATGCGCATGGGCGAGCGGCCGAACGAGCCCATCGAGGTCATCCCCACCGGGTCGACCGCACTCGACGTCGCACTCGGCGTCGGCGGCATCCCCCGCGGCCGCGTGGTGGAGGTGTACGGCCCGGAGTCCTCCGGTAAGACGACCCTCACCCTGCACGCCGTGGCCAACGCCCAGCGGGCCGGTGGCGCGGTGGCCTTCGTGGACGCCGAGCACGCCCTCGACCCGGAGTACGCCAAGAAGCTCGGCGTGGACATCGACAACCTCATCCTGTCCCAGCCGGACAACGGCGAGCAGGCTCTCGAGATCGTCGACATGCTCGTCCGCTCCGGAGCCCTCGATCTGATCGTCATCGACTCCGTCGCCGCCCTGGTGCCGCGCGCGGAGATCGAGGGCGAGATGGGCGACTCGCACGTAGGTCTGCAGGCCCGCCTGATGAGCCAGGCGCTCCGGAAGATCACCAGCGCGCTCAACCAGTCCAAGACCACCGCGATCTTCATCAACCAGCTCCGCGAGAAGATCGGCGTGATGTTCGGCTCCCCGGAGACCACGACCGGTGGCCGCGCGCTGAAGTTCTACGCCTCCGTGCGCATGGACATCCGCCGCATCGAGACCCTCAAGGACGGCACGGACGCGGTGGGCAACCGCACCCGCGTCAAGGTCGTCAAGAACAAGGTCGCGCCCCCCTTCAAGCAGGCCGAGTTCGACATCCTCTACGGCCAGGGCATCAGCCGCGAGGGCGGCCTGATCGACATGGGCGTGGAGCACGGCTTCGTCCGCAAGGCCGGCGCTTGGTACACGTACGAGGGCGACCAGCTCGGCCAGTCAAGAGATCGCCC(SEQ ID NO.6).

[0059] The sequence of primer atpD-230F is: 5'-GAYGAYCCNGARGTNATGAA-3' (SEQ ID NO.7); the sequence of primer atpD-680R is: 5'-CCRTCNGCRTANGCCATCCA-3' (SEQ ID NO.8).

[0060] It should be noted that primers atpD-230F and atpD-680R are degenerate primers designed for the highly conserved region of the bacterial atpD gene. In these primers, Y represents C or T, R represents A or G, and N represents any one of A, T, C, and G.

[0061] The sequence of the atpD gene of strain LY7 is as follows: CTATGCTTGACAGCAATGTCTTCCTCGAGGTCATGTGGGCCGACCCGTCCACGGTCGCCGAGGCCGAGCGCTGGACGATCCACCGCAAGGCCCCGGCCTTCGACCAGCTCGAGTCCAAGACCGAGATGTTCGAGACCGGCCTGAAGGTCGTCGACCTTCTCACCCCGTACGTCAAGGGTGGAAAGATCGGTCTGTTCGGTGGTGCCGGTGTCGGCAAGACCGTGCTGATCCAGGAAATGATCGTCCGTGTGGCCAAGCTGCACGACGGTGTCTCCGTCTTCGCCGGTGTCGGCGAGCGCACCCGTGAGGGCAACGACCTCATGGTCGAGATGGAGGAAGCCGGCGTTCTGGACAAGACCGCGCTGGTCTTCGGCCAGATGGACGAGCCGCCGGGCACGCGTCTGCGCGTGGCCCTGGCCGGTCTGACCATGGCGGAGTACTTCCGCGATGTGCAGAAGCAGGACGTGCTGTTCTTCATCGACAACATCTTCCGCTTCACCCAGGCCGGTTCCGAGGTCTCGACCCTGCTCGGCCGCATGCCCTCCGCGGTGGGCTACCAGCCGAACCTGGCCGACGAGATGGGTCTCCTCCAGGAGCGCATCACCTCGACCCGTGGTCACTCGATCACCTCGATGCAGGCGATCTACGTCCCCGCGGACGACCTGACCGACCCGGCCCCGGCCACCACCTTCGCCCACCTCGACGCGACGACGGTGCTCTCCCGTCCGATCTCGGAGAAGGGCATCTACCCGGCCGTGGACCCGCTGGACTCCACGTCCCGCATCCTGGACCCGCGCTACATCGCGCAGGACCACTACGACGCCGCCATGCGCGTCAAGGGAATCCTGCAGAAGTACAAGGACCTCCAGGACATCATCGCGATCCTCGGCATCGACGAGCTCGGCGAAGAGGACAAGCTCGTCGTGTCCCGTGCAGCGTGCGTGTGGATGCGTCAG (SEQ ID NO.9).

[0062] As shown in Figure 3, strain LY7 clusters in the same clade as Streptomyces alboflavus MDJK44 and Streptomyces alboflavus NRRLB2373, indicating a close phylogenetic relationship. Ultimately, strain LY7 was identified as belonging to Streptomyces alboflavus (S. alboflavus).

[0063] Based on morphological observation, physiological and biochemical index determination, and molecular sequencing and analysis results, LY7 was finally identified as 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.

[0064] Example 2: To further clarify the activity of the strain, the antibacterial spectrum of Streptomyces leucosus LY7 strain obtained from the initial screening, which showed significant antibacterial effects against two soil-borne pathogens of ginseng, was determined.

[0065] The inhibitory effects of the strains on 12 pathogens, namely F. oxysporum, R. solani, Fusarium solani, Phytophthora cactorum, Alternaria alternata, Fusarium verticillioides, Colletotrichum destructivum, Coniothyrium fuckelii, Alternaria alternata, Ilyonectria robusta, Colletotrichum gloeosporioides, and Botrytiscinerea, were determined using the plate two-well confrontation method. (The above-mentioned target pathogens were isolated, identified, preserved, and provided by the Green Prevention and Control Laboratory for Medicinal Plant Diseases of Jilin Agricultural University. Information on the tested pathogens is shown in Table 2.)

[0066] Table 2. Tested pathogenic fungi

[0067] The study found that *Streptomyces leucosus* LY7 exhibited broad-spectrum inhibitory effects against all 12 tested target pathogens (see Figure 4). The inhibitory effects of *Streptomyces leucosus* LY7 on the 12 pathogens are shown in Table 3.

[0068] Table 3. Inhibitory effects of Streptomyces leucovorum LY7 on 12 pathogens.

[0069] As shown in Figure 4, the *Streptomyces leucanthin* strain LY7 exhibited broad-spectrum and significant antifungal 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 leucanthin* strain LY7 demonstrated strong inhibitory activity against multiple plant pathogenic fungi, exhibiting excellent overall antifungal effects and showing promising potential for biocontrol applications.

[0070] Example 3 Creation and Application of Microbial Fertilizer 1. Optimization of Shake Flask Fermentation Conditions of Streptomyces LY7 strain Using single-factor experiments and response surface methodology, the shake flask fermentation conditions of Streptomyces LY7 strain were optimized with the antibacterial activity against F. oxysporum as the response value.

[0071] 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).

[0072] Table 4. Formulas of the basal culture medium used in the test

[0073] 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.

[0074] 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%.

[0075] The nitrogen and carbon sources selected were: yeast powder, beef extract, peptone, soybean flour, potassium nitrate, and urea; the concentrations of the nitrogen and carbon sources selected were: 0%, 1%, 1.5%, 2%, 2.5%, and 3%.

[0076] 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%.

[0077] 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.

[0078] On the other hand, based on the determination of the optimal fermentation medium formulation, single-factor and response surface experiments were also conducted on the environmental conditions (inoculum size, culture temperature, initial pH, bottle size, shaker speed and culture time) of shake flask culture. Finally, the optimal shake flask culture conditions for Streptomyces leucosus strain LY7 were determined to be: pH 7.0, bottle size 75mL / 250mL, 1% inoculum size, 28℃, and cultured at 200rpm for 103 h (Figures 5 and 6).

[0079] 2. Creation and application of microbial fertilizer (1) Fermentation of enoki mushroom substrate The substrate was fermented in accordance with the "Technical Specification for Fermentation of Edible Fungi Residue" (NY / T 3291-2018).

[0080] 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.

[0081] (2) Compatibility evaluation of *Streptomyces leucopsis* LY7 and *Flammulina velutipes* mycelium residue. Preparation of *Flammulina velutipes* mycelium residue extract: The fermented *Flammulina velutipes* mycelium residue was placed in an 80 ℃ oven for dehydration and drying to constant weight. After pulverization, *Flammulina velutipes* mycelium residue powder was obtained and 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. After centrifugation to remove the precipitate, the extract was filtered through a sterile filter membrane to obtain the *Flammulina velutipes* mycelium residue extract.

[0082] 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.

[0083] As shown in Figure 7, the extract of enoki mushroom substrate had no inhibitory effect on the strain, and Streptomyces leucovorum LY7 could be used for the creation of microbial fertilizers.

[0084] (3) Creation of microbial inoculants: The enoki mushroom substrate fermentation product was mixed with Streptomyces oryzae 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.

[0085] 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 antibacterial activity against *F. oxysporum* was determined using the plate confrontation method to screen for the optimal ratio.

[0086] 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 The inhibition rate against Fusarium oxysporum reached 77.14%.

[0087] 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.

[0088] The results in Table 4 and Figure 8 show 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.

[0089] The inhibition rates against mycelial growth of *F. oxysporum* were 79.28–92.79%, against *R. solani* were 69.37–99.10%, against *F. solani* were 50.52–99.48%, against *I. robusta* were 70.31–95.83%, and against *B. cinerea* were 79.69–99.48%.

[0090] Microbial fertilizer extracts based on enoki mushroom substrate showed significant antibacterial activity against Fusarium oxysporum and Fusarium solani (which cause root rot in ginseng), I. robusta (which causes rust rot in ginseng), Botrytis cinerea (which causes gray mold in ginseng), and Rhizoctonia solani (which causes damping-off in ginseng) (Table 5, Figure 8).

[0091] Table 5. Inhibitory effects of different dilutions of bacterial fertilizer extract on the growth of pathogens causing major root diseases in ginseng.

[0092] 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.

[0093] (3) The field application effect evaluation test of microbial fertilizer was conducted in Fusong. A total of 6 treatments were set up, including 1 blank control group (CK) and 5 microbial fertilizer treatment groups.

[0094] 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.

[0095] 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. 2Under the treatment, the ginseng plant height, stem length, stem diameter and other growth indicators showed outstanding performance, and the yield increase effect was significantly higher than other treatments. The overall effect on ginseng growth and yield increase was the best (as shown in Table 6, Table 7, and Figure 9).

[0096] Table 6. Effects of microbial inoculants on the aboveground growth indicators of ginseng.

[0097] 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.

[0098] Table 7. Effects of microbial inoculants on ginseng root growth indicators and yield.

[0099] 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.

[0100] 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, reaching 62.55% against ginseng root diseases (Table 8, Figure 10). In addition, microbial fertilizer effectively regulated the physicochemical properties of ginseng-growing soil and improved enzyme activity at 800 g / m³. 2 The performance of each indicator under the treatment is outstanding (as shown in Tables 9 and 10).

[0101] Table 8. The preventive effect of microbial fertilizer on ginseng root diseases.

[0102] 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.

[0103] Table 9. Effects of microbial inoculants on the physicochemical properties of ginseng rhizosphere soil.

[0104] 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.

[0105] Table 10 Effects of microbial inoculants on enzyme activity in ginseng rhizosphere soil

[0106] 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.

[0107] The results of the above embodiments indicate that the microbial fertilizer prepared by combining *Streptomyces oryzae* strain LY7 with *Flammulina velutipes* spawn can exhibit significant antibacterial activity against *Fusarium oxysporum* and *Fusarium solani* (causing root rot in ginseng), *I. robusta* (causing rust rot in ginseng), *Botrytis cinerea* (causing gray mold in ginseng), and *R. solani* (causing damping-off in ginseng). It significantly promotes ginseng growth and has a significant yield-increasing effect, which is significantly better than that of prebiotic fertilizers and *Flammulina velutipes* spawn. Furthermore, this microbial fertilizer significantly reduces the occurrence of ginseng root rot and can effectively regulate the physicochemical properties of ginseng-growing soil and improve soil enzyme activity.

[0108] 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 leucosus, characterized in that, The strain of Streptomyces alboflavus is named LY7 and classified as Streptomyces alboflavus. 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 application of the *Streptomyces cylindrica* as described in claim 1, the fermentation broth as described in claim 2, or the fermentation broth prepared by the preparation method as described in claim 3 in any one or more of the following: A1. Preparing products for preventing and controlling ginseng diseases; A2. Preventing and controlling ginseng diseases; A3. Promoting ginseng growth; A4. Preparing microbial fertilizers; A5. Regulating soil physicochemical properties; A6. Preparing soil conditioners.

5. The application according to claim 4, characterized in that, The pathogens include one or more of the following: *Fusarium oxysporum*, *Fusarium solani*, *Ilyonectria robusta*, *Botrytis cinerea*, *Rhizoctonia solani*, *Phytophthoracactorum*, and *Alternaria alternata*; 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.

6. The application of the *Streptomyces leucanthin* as described in claim 1, the fermentation broth as described in claim 2, or the fermentation broth prepared by the preparation method as described in claim 3 in any of the following: B1, controlling one or more diseases of tobacco anthracnose, tobacco red spot disease, corn stalk rot, mango anthracnose, and raspberry leaf blight; B2, preparing a product for controlling any one of the pathogens of *Colletotrichum destructivum* (tobacco anthracnose pathogen), *Alternaria alternata* (tobacco red spot pathogen), *Fusarium verticillioides* (corn stalk rot pathogen), *Colletotrichum gloeosporioides* (mango anthracnose pathogen), and *Coniothyrium fuckelii* (raspberry leaf blight pathogen).

7. A method for preparing a microbial fertilizer, characterized in that, The method includes the following steps: mixing the fermentation broth prepared by the Streptomyces flocculation of claim 1, the fermentation broth of claim 2, or the preparation method of claim 3 with the enoki mushroom mycelium fermentation product.

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

9. The application of the microbial fertilizer obtained by the preparation method of claim 7 or the microbial fertilizer of claim 8 in any one or more of the following: C1, prevention and control of ginseng diseases; C2, promotion of ginseng growth; C3, restoration of soil for planting ginseng.

10. The application as described in claim 9, characterized in that, The ginseng diseases mentioned include ginseng root rot, ginseng rust rot, ginseng gray mold, ginseng damping-off, ginseng blight, and ginseng black spot; the growth traits mentioned include ginseng yield and / or plant height.

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