Streptomyces stevensonii nm-1 and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of Streptomyces steviolus. Streptomyces stephensoniae Its applications. The *Streptomyces steffensenii* NM-1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20253020, and its 16S rDNA sequence is shown in SEQ ID NO:1. This strain produces hydrocyanic acid and secretes cellulase, chitinase, protease, and lipase, and can grow in a wide range of environments from pH 5.0 to 12.0 and temperature 10 to 35℃. Plate confrontation experiments showed that this strain has significant inhibitory effects on various plant pathogenic fungi, including *Fusarium wilt* of tomato, *Fusarium graminearum* of wheat, *Anthracnose* of pepper, *Fusarium canker* of walnut, *Fusarium wilt* of cucumber, and *Fusarium root rot* of garlic. Pot experiments confirmed that the fermentation broth of this strain achieved a control efficacy of 74.35% against *Fusarium wilt* of tomato. This invention enriches the strain resource bank and shows promising application prospects in the green control of fungal diseases in crops.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Streptomyces steviolus NM-1 and its applications. Background Technology

[0002] Fusarium wilt of tomato is a disease caused by Fusarium oxysporum tomato-specific strain (Fusarium oxysporum). Fusarium oxysporum f.sp. lycopersici Tomato spores (Fol) are a serious soil-borne disease that can infect tomatoes throughout their entire growth cycle, often leading to acute death of seedlings and concentrated outbreaks during flowering, fruit setting, and fruit enlargement. Currently, control of this disease mainly relies on agricultural management, physical measures, chemical pesticides, and biological control. However, chemical control has problems such as pesticide residues, harm to non-target organisms, induction of pathogen resistance, and disruption of soil microecology; crop rotation is less effective due to the long-term survival of pathogen spores in the soil; and resistant varieties face the risk of resistance degradation and loss due to pathogen mutation. Biocontrol microorganisms are increasingly attracting the attention of agricultural researchers due to their environmental friendliness, green and environmentally friendly nature, and low likelihood of resistance development.

[0003] Streptomyces are Gram-positive bacteria widely distributed in nature. As an important microbial resource, Streptomyces exhibits significant advantages in the biological control of soil-borne diseases due to their strong ability to synthesize antimicrobial active substances, good soil adaptability, and rhizosphere colonization potential. They can synthesize a variety of novel and highly active metabolites, as well as hormones that promote plant growth, glucanases, chitinases, and other substances, showing significant application potential in the green control of plant diseases and pests. Furthermore, the Streptomyces genome contains a large number of gene clusters for the synthesis of secondary metabolites, most of which encode metabolites that have not yet been isolated and identified, possessing immense research value. Despite the abundance of Streptomyces resources and the presence of some strains with antimicrobial activity, their inhibitory activity against Fusarium wilt, the causal agent of tomato wilt, is weak, and they struggle to maintain a sustained control effect in complex soil ecosystems.

[0004] Therefore, exploring new biocontrol bacteria resources, evaluating their control effects on soil-borne diseases such as tomato wilt, and investigating their active metabolites are of great research value for the green control of soil-borne diseases such as tomato wilt. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention screened a *Streptomyces steffusum* NM-1 strain from the rhizosphere soil of healthy tomatoes. This strain produces hydrocyanic acid and secretes cellulase, chitinase, protease, and lipase, and can grow in a wide range of environments including 0-7% NaCl (w / v), pH 5.0-12.0, and temperature 10-35℃. Plate confrontation experiments showed that this strain can inhibit *Fusarium wilt* (the pathogen that causes tomato wilt). Fusarium oxysporum f. sp.lycopersici ), wheat scab ( Fusarium graminearum ), pepper anthracnose bacteria ( Colletotrichum capsici ), Walnut canker pathogen ( Botryosphaeria dothidea ), cucumber wilt pathogen ( Fusarium oxysporum f.sp. cucumerinum ) and garlic root rot fungus ( Fusarium avenaceum This strain can grow against various plant pathogenic fungi, exhibiting a broad antibacterial spectrum. Pot experiments confirmed that the fermentation broth of this strain has a significant control effect on tomato wilt disease. This invention is the first to utilize *Streptomyces steviolense*. Streptomyces stephensoniae NM-1 has been applied to the biological control of plant fungal diseases, providing a new microbial resource with a broad antibacterial spectrum and strong adaptability for the control of plant fungal diseases.

[0006] On one hand, the present invention provides a strain of *Streptomyces stearothermium*, which is named... Streptomyces stephensoniae NM-1.

[0007] Specifically, the *Streptomyces steviolus* was obtained by screening and isolation from the soil of the rhizosphere of healthy tomatoes. The preservation information of the *Streptomyces steviolus* is as follows:

[0008] Strain name: NM-1; Category Name: Streptomyces stephensoniae ; The collection was received by the preservation center on December 29, 2025. Date of issuance of preservation certificate: January 5, 2026; Preservation institution: China Center for Type Culture Collection (CCTCC); Accession number: CCTCC M 20253020.

[0009] Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, China.

[0010] Furthermore, the 16S rDNA sequence of the *Streptomyces steviolus* is shown in SEQ ID NO:1.

[0011] Furthermore, the *Streptomyces steviolus* can produce hydrocyanic acid.

[0012] Furthermore, the *Streptomyces steviolus* can secrete one or more of cellulase, chitinase, protease, and lipase.

[0013] Furthermore, the *Streptomyces steviolus* can grow in an environment with a pH value of 5.0-12.0.

[0014] Furthermore, the *Streptomyces steviolus* can grow at temperatures ranging from 10°C to 35°C.

[0015] In another aspect, a microbial agent is also provided, which contains *Streptomyces steviolense* as described in this invention.

[0016] Furthermore, this invention also provides the application of *Streptomyces steviolus* or microbial agents in inhibiting the growth of pathogens, wherein the pathogen is *Fusarium wiltii* (the causal agent of tomato wilt). Fusarium oxysporum f. sp. lycopersici ), wheat scab ( Fusarium graminearum ), pepper anthracnose bacteria ( Colletotrichum capsici ), Walnut canker pathogen ( Botryosphaeria dothidea ), cucumber wilt pathogen ( Fusarium oxysporum f.sp. cucumerinum ) and garlic root rot fungus ( Fusarium avenaceum One or more of the following.

[0017] On the other hand, a microbial agent is also provided, the microbial agent containing fermentation broth of Streptomyces steffensen and / or cell-free filtrate of Streptomyces steffensen fermentation broth and / or extract of Streptomyces steffensen fermentation broth supernatant, wherein the Streptomyces steffensen has the preservation number CCTCC M 20253020.

[0018] Finally, the application of the microbial agent described in this invention in the control of tomato wilt disease is also provided, using *Streptomyces steviolense*. Streptomyces stephensoniae NM-1 fermentation broth was used to drench the roots of tomato seedlings. The tomato wilt disease was caused by Fusarium oxysporum tomato-specific strain (… Fusarium oxysporum f. sp. lycopersici (Induced by)

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) In this invention, a strain of Streptomyces steviolus NM-1 was obtained from the soil of the rhizosphere of healthy tomatoes and named Streptomyces stephensoniae NM-1, the first report of Streptomyces steviolense. Streptomyces stephensoniae The application of this strain in the biological control of plant fungal diseases has enriched the resources of microbial strains. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20253020, and its 16S rDNA sequence is shown in SEQ ID NO:1. Plate confrontation experiments showed that this strain exhibited significant antagonistic activity against various plant pathogenic fungi, including *Fusarium wilt* of tomato, *Fusarium graminearum* of wheat, *Anthracnose* of pepper, *Pseudomonas canker* of walnut, *Fusarium wilt* of cucumber, and *Pseudomonas root rot* of garlic, overcoming the limitation of some biocontrol bacteria targeting only a single pathogen.

[0020] (2) This strain has multiple biocontrol mechanisms. It can not only directly inhibit pathogens by producing antibacterial active substances, but also degrade the cell walls of pathogens or compete for nutrient space by secreting cellulase, chitinase, etc., forming multiple disease prevention barriers and making the effect more stable.

[0021] (3) This strain has strong environmental adaptability and is tolerant to salt and alkali and certain temperature changes, which helps it to successfully colonize in complex rhizosphere soil environments and exert its biocontrol function for a long time. In addition, the live cells, fermentation broth, cell-free filtrate and extracts of this strain are all active and have the potential for the development of various formulations, which provides an important foundation for the application of this biocontrol strain.

[0022] (4) The results of the pot experiment showed that the application of the fermentation liquid of this strain had a control effect of 74.35% on tomato wilt. Attached Figure Description

[0023] Figure 1 Streptomyces Stephenson Streptomyces stephensoniae The in-plate antagonistic effect of NM-1 on Fol; where a is a control image of Fol inoculated alone; b is an in-plate confrontation image of inoculated strain NM-1 and Fol.

[0024] Figure 2 Streptomyces Stephenson Streptomyces stephensoniae Morphological characteristics of NM-1; where a represents Streptomyces steviolense. Streptomyces stephensoniae Growth of NM-1 on PDA medium; b is Streptomyces steffensen. Streptomyces stephensoniae The morphology of NM-1 on PDA medium; c represents *Streptomyces steffensonii*. Streptomyces stephensoniae Morphology of NM-1 spores under a scanning electron microscope.

[0025] Figure 3 Streptomyces Stephenson Streptomyces stephensoniae Phylogenetic tree of NM-1.

[0026] Figure 4 Streptomyces Stephenson Streptomyces stephensoniae The whole genome map of NM-1.

[0027] Figure 5 Streptomyces Stephenson at different volume fractions Streptomyces stephensoniae The figure shows the results of the NM-1 cell-free fermentation filtrate's inhibition of Fol mycelial growth. In this figure, 'a' represents different volume fractions of *Streptomyces steviolus*. Streptomyces stephensoniae Fol mycelial growth diagram after treatment with NM-1 cell-free fermentation filtrate; b shows different volume fractions of Streptomyces steffensonii. Streptomyces stephensoniae Statistical results of the inhibition rate of Fol mycelia by NM-1 cell-free fermentation filtrate.

[0028] Figure 6 Streptomyces Stephenson at different volume fractions Streptomyces stephensoniae The effect of NM-1 cell-free fermentation filtrate on Fol spore germination is shown in the figure. Where 'a' represents different volume fractions of *Streptomyces steviolense*. Streptomyces stephensoniae Microscopic images of Fol spore germination after treatment with NM-1 cell-free fermentation filtrate; b shows different volume fractions of Streptomyces steviolus. Streptomyces stephensoniae Figure 1 shows the germination rate of Fol spores after treatment with NM-1 cell-free fermentation filtrate.

[0029] Figure 7 Different concentrations of Streptomyces Stephenson Streptomyces stephensoniae The effect of crude extract from NM-1 fermentation broth supernatant on Fol mycelial growth is shown in the figure. Figure a represents different concentrations of *Streptomyces steviolus*. Streptomyces stephensoniae Fol mycelial growth diagram after treatment of crude extract from NM-1 fermentation broth supernatant; b shows different concentrations of Streptomyces steffensonii. Streptomyces stephensoniae Statistical results of Fol mycelial inhibition rate in the crude extract treatment group of NM-1 fermentation broth supernatant.

[0030] Figure 8 Streptomyces Stephenson Streptomyces stephensoniae The efficacy of NM-1 fermentation broth in preventing tomato wilt in potted plants. Detailed Implementation

[0031] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0032] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0033] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0034] Example 1 This embodiment describes the isolation and screening of Streptomyces Stephenson and the detection of its antibacterial spectrum.

[0035] 1.1 Isolation and purification of strains Dissolve 1g of soil powder from the rhizosphere of healthy tomatoes in 10mL of sterile water, shake at 180rpm for 1h in a constant temperature shaker, allow to settle, and then take the supernatant and serially dilute it with sterile water to a 10⁻⁶ solution. -2 10 -3 and 10 -4Soil suspensions of different dilutions were prepared. 100 μL of each soil suspension was spread onto GS-1 and PDA media for isolation and culture, with three plates for each concentration. The media were incubated at 28℃ for 7 days, and single colonies of actinomycetes were collected. Based on colony morphology and color differences, single colonies were isolated and cultured on PDA and GS media until sporulation. 5 mL of sterile distilled water was added to the culture dish, and spores were gently scraped from the surface of the medium using a sterile cotton swab to collect the liquid. The liquid was then filtered through a sterile injection filter lined with absorbent cotton to remove mycelium and residual medium, yielding a pure bacterial spore solution. The pure bacterial spore solution was stored in 25% glycerol tubes at -80℃. The PDA medium consisted of 200 g peeled potatoes, 20 g glucose, 15 g agar powder, and 1 L distilled water. The GS-1 culture medium consists of 20g soluble starch, 1.0g KNO3, 0.5g KH2PO4, 0.5g NaCl, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 15g agar powder, and 1L distilled water.

[0036] 1.2 Plate confrontation test was used to screen strains that inhibited the pathogen of tomato wilt. Inoculate the center of the PDA medium with the pathogen of tomato wilt ( Fusarium oxysporum f.sp. lycopersici For the *Fol* mycelium, 1.1 μL of purified strain was inoculated at 1.5 cm to the left and right of the edge of the petri dish. A medium inoculated only with *Fol* mycelium was used as a control. The dishes were incubated at 28°C until the control mycelium fully colonized the petri dishes. The width of the inhibition zone in each treatment group was measured. Each treatment was repeated three times. The results are shown in Table 1 and [Table data would be inserted here]. Figure 1 As shown. A strain with antagonistic activity against the pathogen of tomato wilt was obtained through screening and labeled as NM-1.

[0037] Table 1. Antimicrobial effect of strain NM-1 against the pathogen of tomato wilt.

[0038] 1.3 The antibacterial spectrum of strain NM-1 was tested using a plate confrontation test. Wheat scab fungus was inoculated into the center of the PDA medium. Fusarium graminearum ), pepper anthracnose bacteria ( Colletotrichum capsici ), Walnut canker pathogen ( Botryosphaeria dothidea ), cucumber wilt pathogen ( Fusarium oxysporum f.sp. cucumerinum ) and garlic root rot fungus ( Fusarium avenaceumStrain NM-1 was inoculated on both sides of the edge of the petri dish. Controls were prepared by inoculating media containing only *Fusarium graminearum* (wheat scab), only *Anthracnose causal agent* (pepper anthracnose), only *Walnut canker* (walnut rot), only *Fusarium wilt* (cucumber rot), and only *Radix et rotae* (garlic root rot). The media were incubated at 28°C until the control media completely covered the petri dish. The width of the inhibition zone in each treatment group was measured, with each treatment repeated three times. The results are shown in Table 2.

[0039] Table 2. Antibacterial effects of strain NM-1 against other pathogens.

[0040] As shown in Table 2, strain NM-1 not only has antibacterial activity against Fusarium wilt of tomato, but also has significant antibacterial activity against Fusarium wilt of wheat, Anthracnose of pepper, Fusarium canker of walnut, Fusarium wilt of cucumber and root rot of garlic.

[0041] Example 2 This example demonstrates the identification of strain NM-1.

[0042] 2.1 Morphological Characteristics Identification The strain NM-1 obtained in Example 1 was streaked onto ISP1-7 series medium and 2CMY, MS, GS, PDA and AS-1 medium and cultured at 28℃ for 7 days. The culture characteristics of strain NM-1 were recorded and observed. After 14 days of culture, 5mm × 5mm bacterial blocks were cut from PDA medium, fixed and dehydrated, critically dried, sputter-coated with gold, and observed under a scanning electron microscope. The morphological identification results are as follows. Figure 2 As shown in Table 3, the culture medium formulations involved are shown in Table 4. Figure 2 Figures a and b show that strain NM-1 appears as white flocculent mycelium on PDA medium, with well-developed aerial hyphae and pale yellow mycelium in the substrate. Figure 2 As shown in the image, when observed under a scanning electron microscope, strain NM-1 has a large number of spores, which are elliptical to cylindrical, with a smooth surface, a size of about 1 μm, and beaded spore chains with long spore chains.

[0043] Table 3. Observation results of culture characteristics of strain NM-1 Note: "++" indicates good growth; "+" indicates weak growth.

[0044] Table 4. Composition of culture medium for morphological identification of strain NM-1 2.2 Physiological and biochemical characteristics of strain NM-1 The physiological and biochemical characteristics of strain NM-1 were determined using conventional methods, and the results are shown in Table 5. Simultaneously, the salt tolerance, pH tolerance, and temperature tolerance of strain NM-1 were determined. Specifically, strain NM-1 was streaked onto PDA medium with different NaCl contents (1-10% NaCl M / V, with 1% intervals), and incubated at 28℃ for 10 days. The growth of the test strain was then observed and recorded. Strain NM-1 was also streaked onto PDA medium and incubated at different temperatures (5-50℃, with 5℃ intervals) for 10 days, and the growth of the test strain was then observed and recorded. In a sterile environment, sterile PDB culture medium was prepared to different pH values ​​(pH 4-10, with 1-degree intervals), and the same amount of strain NM-1 was inoculated. After incubation with shaking at a constant temperature for 7 days, the growth of the test strain was observed and recorded, and the results were statistically analyzed. The statistical results are shown in Table 5.

[0045] As shown in Table 5, strain NM-1 can utilize most carbon and nitrogen sources and has salt and alkali tolerance.

[0046] Table 5. Physiological and biochemical indicators of strain NM-1 Note: "-" indicates a negative reaction; "+" indicates a positive reaction.

[0047] 2.3 Molecular biological identification of strain NM-1 Genomic DNA was extracted from strain NM-1, and its 16S rDNA sequence was amplified using universal primers 27F and 1492R. After verification by agarose gel electrophoresis, the 16S rRNA gene sequence was obtained through sequencing. (The *Streptomyces stenosus* strain was described.) Streptomyces stephensoniae The 16S rRNA gene sequence of NM-1 is shown in SEQ ID NO:1. The *Streptomyces steviolus* species... Streptomyces stephensoniae The specific 16S rRNA gene sequence of NM-1 is as follows:

[0048] Streptomyces Stephenson Streptomyces stephensoniae The NM-1 whole-genome sequencing results were analyzed, and a phylogenetic tree was constructed using the Type Strain Genome Server website, such as... Figure 3 As shown, the results identified strain NM-1 as *Streptomyces steviolense*. Streptomyces stephensoniae Streptomyces Stephenson Streptomyces stephensoniae The whole genome map of NM-1 is as follows Figure 4 As shown. By Figure 4 It is known that *Streptomyces steviolus* Streptomyces stephensoniae The NM-1 genome is 7,928,148 bp in length with a GC content of 71.91%. Gene function annotation of the entire genome was performed using the NR, COG, KEGG, and GO databases. A total of 6,838 coding genes were predicted for this strain, accounting for 87.96% of the total genome length. Prediction of coding genes, repetitive sequences, and non-coding RNAs was also performed to obtain the *Streptomyces stenosus* genome. Streptomyces stephensoniae The NM-1 genome is composed of 65 tRNAs, 6 5S rRNAs, 6 16S rRNAs, 6 23S rRNAs, and 62 sRNAs.

[0049] Example 3 This example uses *Streptomyces steviolus*. Streptomyces stephensoniae Prediction of NM-1 genome-wide secondary metabolic biosynthesis gene clusters.

[0050] Predicting Streptomyces Stephenson using the antiSMASH database Streptomyces stephensoniae The metabolites of NM-1 and the results of antiSMASH database analysis are shown in Table 6.

[0051] Table 6. Prediction results of secondary metabolic biosynthesis gene clusters in the whole genome of strain NM-1

[0052] As shown in Table 6, *Streptomyces steviolense* Streptomyces stephensoniae NM-1 contains 34 secondary metabolic gene clusters, including nucleosides, polyketides, non-ribosomal peptides, and lanothiopeptides. Nine of these clusters show 100% similarity to known gene clusters, indicating that *Streptomyces steviolense*... Streptomyces stephensoniae NM-1 is highly likely to produce these substances. Furthermore, there are gene clusters with similarity below 30% or zero, indicating that *Streptomyces steviolus* is likely involved. Streptomyces stephensoniae NM-1 has the ability to produce some unknown antibacterial compounds.

[0053] Example 4 This example uses *Streptomyces steviolus*. Streptomyces stephensoniae Detection of secondary metabolites of NM-1.

[0054] Detection of Streptomyces Stephenson using conventional methods Streptomyces stephensoniae Secondary metabolites of NM-1.

[0055] Protease production capacity assay: 20 μL of Streptomyces Stephenson was added to the test kit. Streptomyces stephensoniae NM-1 was inoculated into protease detection medium and incubated at 28°C for 7 days. The appearance of a clear zone around the colony indicates that it has the ability to produce protease.

[0056] Phosphate-solubilizing ability test: Streptomyces Stephenson Streptomyces stephensoniae NM-1 was inoculated into an inorganic phosphorus detection medium and cultured at 28°C for 7 days. If a transparent phosphorus-solubilizing zone was formed, it indicated that the plant had the ability to dissolve inorganic phosphorus.

[0057] Ferrophilic secretion assay: Streptomyces Stephenson Streptomyces stephensoniae NM-1 was inoculated into CAS medium and cultured at 28°C for 7 days. The appearance of a clear zone indicates that the strain can secrete siderophiles.

[0058] HCN production detection: Prepare HCN detection medium according to the formula, and streak inoculate with Streptomyces streaksburger. Streptomyces stephensoniae After NM-1 was cultured at 28℃ for 7 days, the color around the colonies was observed. The HCN production capacity of the test strain was divided into three levels according to the color change: orange, red, and brown, representing weak, moderate, and strong, respectively.

[0059] IAA production capacity test: Prepare DF culture medium and Salkowski reagent according to the formula, and add 30 μL of Streptomyces Stephenson. Streptomyces stephensoniae The spore suspension of NM-1 was inoculated into a 10 mL centrifuge tube containing 5 mL of DF culture medium and cultured at 28 °C and 180 rpm for 7 days with constant temperature shaking. After centrifugation at 5000 rpm for 10 min, the supernatant was taken and mixed with the prepared Salkowski reagent at a ratio of 1:2. The solution turned red, indicating that the strain had the ability to produce IAA.

[0060] Detection of β-1-3-glucanase production: Streptomyces Stephenson Streptomyces stephensoniae After inoculating the NM-1 spore suspension onto the test medium and incubating at 28°C for 7 days, the presence of a clear transparent zone indicates that the strain can secrete β-1-3 glucanase.

[0061] ACC deaminase production detection: Streptomyces Stephenson Streptomyces stephensoniaeThe NM-1 strain was streaked onto DF solid medium containing ACC and incubated at 28°C for 7 days. If the strain could grow normally, it indicated that the strain could produce ACC deaminase.

[0062] Lipase production detection: inoculation with Streptomyces Stephenson Streptomyces stephensoniae The NM-1 strain was cultured on lipase detection medium at 28°C for 7 days. The appearance of a clear or white halo around the colony indicates that it can secrete lipase.

[0063] Cellulase production detection: Prepare CMC medium and add Streptomyces steviolus to the medium. Streptomyces stephensoniae After inoculating the NM-1 strain onto CMC medium and incubating at 28°C for 7 days, stain with 1 mg / mL Congo red for 10-15 min, then decolorize with 1 M NaCl solution for 1 h. If a clear transparent zone appears around the bacterial block, it proves that the strain can secrete cellulase, and the larger the transparent zone, the stronger the ability to produce cellulase.

[0064] Chitinase production capacity assay: Streptomyces Stephenson Streptomyces stephensoniae When NM-1 strain is inoculated onto chitinase medium and cultured at 28°C for 7 days, the appearance of a clear transparent zone indicates that the strain has the ability to secrete chitinase.

[0065] Nitrogen fixation capacity test: Streptomyces Stephenson was inoculated onto nitrogen-fixing medium. Streptomyces stephensoniae If the NM-1 spore suspension can be cultured at 28°C for 7 days, and the strain can grow on the culture medium, it indicates that the strain has nitrogen-fixing ability.

[0066] Streptomyces Stephenson Streptomyces stephensoniae The results of the detection of secondary metabolites of NM-1 are shown in Table 7, and the culture medium used for the detection of secondary metabolites is shown in Table 8. The results show that *Streptomyces steviolense* Streptomyces stephensoniae NM-1 can produce proteases, cellulases, lipases, and chitinases.

[0067] Table 7 Results of the ability of strain NM-1 to produce secondary metabolites

[0068] Note: "-" indicates a negative reaction; "+" indicates a positive reaction.

[0069] Table 8. Culture media for secondary metabolite detection

[0070] Example 5 This embodiment is for evaluating *Streptomyces Stephenson*. Streptomyces stephensoniaeNM-1 cell-free fermentation filtrate against tomato wilt pathogen ( Fusarium oxysporum f. sp. lycopersici The inhibitory effect of Fol.

[0071] 5.1 Assessment of Streptomyces Stephenson Streptomyces stephensoniae Inhibitory effect of NM-1 cell-free fermentation filtrate on Fol bacteria.

[0072] Streptomyces Stephenson grown on PDA plates for 7 days Streptomyces stephensoniae NM-1 bacterial blocks were inoculated into AS-1 medium and cultured at 28°C and 180 rpm for 7 days. The fermentation broth was collected, centrifuged at 10,000 rpm for 10 min, and then filtered through a 0.22 μm bacterial filter to obtain *Streptomyces steffensenii*. Streptomyces stephensoniae Cell-free fermentation filtrate of NM-1. Streptomyces Stephenson Streptomyces stephensoniae Cell-free fermentation filtrate of NM-1 was added to PDA medium to be coagulated and mixed well to prepare Streptomyces Stephenson-containing cultures. Streptomyces stephensoniae NM-1 cell-free fermentation filtrate plates with fractions of 2%, 5%, 10%, and 20% were prepared. A Fol mycelial cake was inoculated in the center of the medium to ensure it was free of *Streptomyces steviolense*. Streptomyces stephensoniae Blank PDA plates containing the cell-free fermentation filtrate of NM-1 were used as controls and incubated at 28°C until the control mycelia completely covered the entire plate. The diameter of the colonies in each treatment was measured using the cross-hatching method, and the mycelial growth inhibition rate was calculated. The results are as follows: Figure 5 As shown, different volume fractions of Streptomyces Stephenson Streptomyces stephensoniae The cell-free fermentation filtrate of NM-1 inhibited the mycelial growth of *Fol*, and the inhibition rate increased with *Streptomyces steviolense*. Streptomyces stephensoniae The cell-free fermentation filtrate fraction of NM-1 increases with the increase of the cell-free fermentation filtrate fraction.

[0073] 5.2 Assessment of Streptomyces Stephenson Streptomyces stephensoniae Inhibitory effect of NM-1 cell-free fermentation filtrate on Fol spore germination.

[0074] Following the method in 5.1, *Streptomyces steviolense* was... Streptomyces stephensoniae NM-1 cell-free fermentation filtrate was mixed with PDB medium to prepare Streptomyces steviolium Stephenson. Streptomyces stephensoniae NM-1 cell-free fermentation filtrates were prepared with fractions of 2%, 5%, 10%, 20%, and 50%. Fol mycelial cakes were inoculated into PDB medium and cultured at 28°C and 180 rpm for 48 hours. The resulting Fol spore suspension was obtained by filtration through sterile filter paper and adjusted to a concentration of 1 × 10⁻⁶ using a hemocytometer. 7 Spores / mL, add 1mL of prepared culture medium of different concentrations to a 2mL centrifuge tube, excluding Streptomyces Stephenson. Streptomyces stephensoniaeThe NM-1 cell-free fermentation filtrate was used as a control in PDB medium. After incubation at 28℃ and 180 rpm for 10 h, samples were taken and spore germination was observed under a microscope. Spore germination data for each treatment were statistically analyzed, and the spore germination inhibition rate was calculated. Results are as follows: Figure 6 As shown, different volume fractions of Streptomyces Stephenson Streptomyces stephensoniae NM-1 cell-free fermentation filtrate can delay or inhibit the spore germination of *Streptomyces steviolus*. Streptomyces stephensoniae When the NM-1 cell-free fermentation filtrate fraction was 5%, the germination inhibition rate of Fol spores reached 50%, and when *Streptomyces steviolus* was... Streptomyces stephensoniae When the NM-1 cell-free fermentation filtrate fraction was 10%, the germination rate of Fol spores was only 9.39%.

[0075] Example 6 This embodiment is for evaluating *Streptomyces Stephenson*. Streptomyces stephensoniae Inhibitory effect of crude extract of NM-1 fermentation broth supernatant on Fol mycelial growth.

[0076] Streptomyces Stephenson Streptomyces stephensoniae NM-1 mycelial cakes were inoculated into AS-1 medium and cultured at 28℃ and 180 rpm for 7 days. The fermentation broth was then collected, centrifuged at 10000 rpm for 10 min, and the supernatant was taken to obtain *Streptomyces steffensonii*. Streptomyces stephensoniae The supernatant of NM-1 fermentation broth contains Streptomyces steviolus. Streptomyces stephensoniae The supernatant of the fermentation broth of NM-1 was extracted with ethyl acetate at a volume ratio of 1:1. The organic phase was collected and evaporated to dryness using a rotary evaporator (40℃). The extract was collected, weighed, and dissolved in methanol to prepare a 50 mg / mL solution, yielding *Streptomyces steviolus*. Streptomyces stephensoniae Crude extract of fermentation broth supernatant of NM-1.

[0077] Add Streptomyces Stephenson to PDA medium Streptomyces stephensoniae The crude extract of the fermentation broth supernatant of NM-1 was prepared as Streptomyces steviolus. Streptomyces stephensoniae NM-1 fermentation broth supernatant crude extract final concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL were prepared into plates containing the extract. These plates were inoculated with Fol culture according to the above method. After 7 days, mycelial growth was observed, colony diameter was measured, mycelial growth inhibition rate was calculated, and EC50 was calculated using DPS software. 50 Value. Result as follows Figure 7 As shown, Streptomyces Stephenson Streptomyces stephensoniae The crude extract of the fermentation broth supernatant of NM-1 inhibited the mycelial growth of *Streptomyces stenosus* in a dose-dependent manner. Calculations showed that *Streptomyces stenosus*...Streptomyces stephensoniae EC of NM-1 fermentation broth supernatant crude extract 50 The value was 45.91 μg / mL.

[0078] Example 7 This embodiment is for evaluating *Streptomyces Stephenson*. Streptomyces stephensoniae NM-1's efficacy in preventing tomato wilt in potted plants.

[0079] Streptomyces Stephenson grown on PDA plates for 7 days Streptomyces stephensoniae NM-1 bacterial blocks were inoculated into PDB medium and cultured at 28°C and 180 rpm for 7 days. The fermentation broth was then collected for later use.

[0080] After disinfecting the surface of tomato seeds, sow them in seedling trays. Once the seedlings reach the 2-3 leaf stage, transplant them into small flowerpots, and then treat with Streptomyces steviolus. Streptomyces stephensoniae Root drenching with NM-1 fermentation broth, 20 mL per plant, followed by root inoculation with Fol spore suspension (10) 3 days later. 7 (Spores / mL), 10mL per pot.

[0081] Prepare an inoculation solution of Fol spores (10) 7 Treatment with spores / mL and inoculation with Streptomyces steffensen Streptomyces stephensoniae NM-1 fermentation broth and Fol spore suspension (10 7 Spores / mL were treated with either spores or water, with 15 pots per treatment. After culturing at 28℃ for 30 days, disease incidence was observed, and the disease index and control effect were calculated. The results are shown in Table 9. Figure 8 As shown.

[0082] Table 9. Results of the control efficacy test of strain NM-1 against tomato wilt disease.

[0083] Table 9 shows that the disease index for Fol-only treatment was 65.00, while the disease index for Streptomyces Stephenson treatment was higher. Streptomyces stephensoniae The disease index after NM-1 treatment was 16.67, indicating *Streptomyces stenosus*. Streptomyces stephensoniae NM-1 achieved a 74.35% control efficacy against tomato wilt in potted tomatoes, indicating that *Streptomyces steviolense*... Streptomyces stephensoniae NM-1 can be used for the control of tomato wilt disease. This invention provides more biological resources for the control of tomato wilt disease.

[0084] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of *Streptomyces steviolense*, characterized in that, The Streptomyces Stephenson was named Streptomyces stephensoniae NM-1 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC M 20253020.

2. The *Streptomyces steviolus* according to claim 1, characterized in that, The 16S rDNA sequence of the Streptomyces Stephenson is shown in SEQ ID NO:

1.

3. The *Streptomyces steviolense* according to claim 1 or 2, characterized in that, The Streptomyces Stephenson species can produce hydrogen cyanide.

4. The *Streptomyces steviolense* according to claim 1 or 2, characterized in that, The Streptomyces Stephenson can secrete one or more of cellulase, chitinase, protease, and lipase.

5. The *Streptomyces steviolense* according to claim 1 or 2, characterized in that, The Streptomyces Stephenson can grow in an environment with a pH of 5.0-12.

0.

6. The *Streptomyces steviolus* according to claim 1 or 2, characterized in that, The Streptomyces Stephenson strain can grow at temperatures ranging from 10°C to 35°C.

7. A microbial inoculant, characterized in that, The microbial agent contains *Streptomyces steviolense* as described in claim 1.

8. The application of *Streptomyces stenosus* as described in claim 1 or the microbial agent as described in claim 7 in inhibiting the growth of pathogenic bacteria, characterized in that... The pathogen is *Fusarium wilt* (Cucumber wilt pathogen). Fusarium oxysporum f.sp. lycopersici ), wheat scab ( Fusarium graminearum ), pepper anthracnose bacteria ( Colletotrichum capsici ), Walnut canker pathogen ( Botryosphaeria dothidea ), cucumber wilt pathogen ( Fusarium oxysporum f.sp. cucumerinum ) and garlic root rot fungus ( Fusarium avenaceum One or more of the following.

9. A microbial agent, characterized in that, The microbial agent contains fermentation broth of Streptomyces stearens and / or cell-free filtrate of Streptomyces stearens fermentation broth and / or extract of Streptomyces stearens fermentation broth supernatant, wherein the Streptomyces stearens has the preservation number CCTCC M 20253020.

10. The application of the microbial agent according to claim 9 in the control of tomato wilt disease, characterized in that, Tomato seedlings were treated with root irrigation using *Streptomyces steviolus* fermentation broth. The tomato wilt disease was caused by *Fusarium oxysporum* tomato-specific strain (…). Fusarium oxysporum f. sp. lycopersici (Induced by)

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