Streptomyces murinus FLWZ-0001 and application thereof in prevention and treatment of crop fungal and oomycete diseases

Abstract

The invention discloses streptomyces murinus FLWZ-0001 and application of the streptomyces murinus FLWZ-0001 in prevention and treatment of fungal and oomycete diseases of crops. The streptomyces murinus FLWZ-0001 is preserved in the China Center for Type Culture Collection on September 5, 2024, and the preservation number of the streptomyces murinus FLWZ-0001 is CCTCC (China Center for Type Culture Collection) NO: M20241923. The strain and the fermentation liquor thereof have a remarkable inhibition effect on various plant pathogenic bacteria, can efficiently inhibit fungal pathogenic bacteria such as sclerotinia sclerotiorum, botrytis cinerea and penicillium digitatum, and can also efficiently inhibit phytophthora capsici and phytophthora nicotianae belonging to oomycetes. Pot experiments show that fermentation liquor of the strain has excellent prevention and treatment effects on multiple crop diseases such as sclerotinia rot of colza, black shank of colza, phytophthora blight of pepper, gray mold and the like. The invention provides a brand new microbial resource for developing a broad-spectrum, efficient and environment-friendly multi-target biopesticide, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of plant disease biological control technology, specifically to the *Streptomyces murinus* strain FLWZ-0001 and its application in controlling fungal and oomycete diseases of crops. More specifically, it relates to a novel *Streptomyces murinus* strain FLWZ-0001 with broad-spectrum and highly effective control over both fungal and oomycete diseases of crops, its fermentation broth, and its application in controlling various plant diseases such as sclerotinia rot in rapeseed, black shank in rapeseed, blight in pepper, gray mold, Phytophthora blight in tobacco, and postharvest diseases of citrus. Background Technology

[0002] Rapeseed and chili peppers are important economic crops in my country, playing a crucial role in agricultural production. However, during their growth and development, they are often threatened by various pathogens, leading to severe yield losses and quality declines. Among them, sclerotinia sclerotiorum rot and black shank rot are two devastating diseases affecting rapeseed production, causing yield reductions or even total crop failure. Meanwhile, Phytophthora capsici blight and Botrytis cinerea gray mold also pose significant threats to the chili pepper industry. These diseases often occur in combination, making control extremely difficult.

[0003] Currently, the control of these diseases mainly relies on chemical fungicides. However, the long-term and excessive use of chemical pesticides not only easily leads to drug resistance in pathogens and increases control costs, but also causes problems such as excessive pesticide residues and environmental pollution, which does not meet the requirements of green and sustainable development in modern agriculture. Therefore, developing environmentally friendly, safe, and efficient biological control technologies has become a research hotspot in the field of plant protection.

[0004] Utilizing beneficial microorganisms and their metabolites to control plant diseases is one of the important directions of biological control. Currently, reported biocontrol bacteria mainly include *Trichoderma* spp., *Bacillus* spp., and *Pseudomonas* spp. However, existing biocontrol bacteria generally suffer from drawbacks such as narrow control spectrum and unstable efficacy. For example, *Trichoderma* has some efficacy against sclerotinia stem rot, but poor efficacy against black shank; *Bacillus* can inhibit some soil-borne diseases, but its control effect on airborne diseases (such as gray mold) is limited. To date, there are no reports of single microbial strains that can simultaneously and effectively control sclerotinia stem rot and black shank in rapeseed, and blight and gray mold in pepper.

[0005] Streptomyces spp. is an important group of biocontrol actinomycetes that produce abundant secondary metabolites and possess broad-spectrum antibacterial activity. Among them, Streptomyces murinus, a species within this genus, has shown biocontrol potential in some strains. For example, Chinese patent document CN104726362A discloses a Streptomyces murinus strain FXQ-5B isolated from mangrove soil. This strain exhibits significant antagonistic activity against Pestalotiopsis microspora, the causal agent of tea leaf blight, and also shows some inhibitory effect on the pathogens of various fungal diseases, such as Fusarium oxysporum. However, research on this strain and related studies has mainly focused on the control of diseases of Camellia oleifera and some fungal diseases, without addressing its control effects on major diseases of crops such as rapeseed and pepper, especially on diseases caused by Phytophthora spp., which belongs to the Oomycota phylum (such as pepper blight and tobacco blight). Its application scope remains limited.

[0006] Therefore, current technologies lack a broad-spectrum biocontrol strain capable of simultaneously and efficiently controlling fungal diseases (such as sclerotinia rot, black shank, and gray mold) and oomycete diseases (such as blight) on multiple crops, including rapeseed and pepper. Thus, discovering a new strain of *Streptomyces griseus* with broad-spectrum antifungal activity, high efficacy against both fungi and oomycetes, and strong environmental adaptability is of significant theoretical and practical value for developing multi-target biological pesticides and solving practical disease problems in agricultural production. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide a novel strain of *Streptomyces griseus* with broad-spectrum and highly effective control of both fungal and oomycete diseases in crops, and its application. This novel strain of *Streptomyces griseus* (named FLWZ-0001) was isolated from the rhizosphere soil of rapeseed. *Streptomyces griseus* FLWZ-0001 and its fermentation broth not only exhibit significant inhibitory effects on various fungal pathogens such as *Sclerotinia sclerotiorum* and *Botrytis cinerea*, but also unexpectedly demonstrate highly effective inhibition of *Phytophthora capsici* and *Phytophthora nicotianae*, belonging to the phylum Oomycota. This enables effective control of various crop diseases, including sclerotinia rot in rapeseed, black shank in rapeseed, blight in peppers, gray mold, blight in tobacco, and postharvest diseases of citrus.

[0008] Therefore, this application provides the following technical solution:

[0009] In the first aspect, this application provides a strain of Streptomyces murinus, which was deposited at the China Center for Type Culture Collection on September 5, 2024, with the following classification and name: Streptomyces murinus FLWZ-0001; accession number: CCTCC NO: M20241923; location: Wuhan University, Wuhan, China.

[0010] Secondly, this application provides a liquid fermentation broth of *Streptomyces flavonoids* FLWZ-0001 as described in the first aspect, which is prepared by a method including the following steps:

[0011] (1) Seed culture: FLWZ-0001 of Streptomyces gravidarum was inoculated into seed culture medium and cultured at 28 °C and 150 r / min for 4 days to obtain seed culture;

[0012] (2) Liquid fermentation: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10%, and culture at 28 °C and 150 r / min for 7 days to obtain the fermentation liquid.

[0013] Thirdly, this application provides the use of Streptomyces flavonoids FLWZ-0001 as described in the first aspect or the liquid fermentation broth as described in the second aspect in the preparation of microbial agents for the prevention and control of plant diseases.

[0014] In some embodiments, the plant disease is a disease caused by fungal pathogens and / or a disease caused by oomycete pathogens.

[0015] In some embodiments, the fungal pathogens include at least one of *Sclerotinias clerotiorum*, *Botrytis cinerea*, *Leptosphaeria maculans*, *Penicillium digitatum*, *Penicillium italicum*, *Colletotrichum gloeosporioides*, *Alternaria solani*, *Fusarium graminearum*, *Fusarium verticillioides*, *Peronophythora litchii*, *Alternaria gaisen*, and *Pestalotiopsis theae*; the oomycete pathogens include at least one of *Phytophthora capsici* and *Phytophthora nicotianae*.

[0016] In some embodiments, the plant disease is at least one of the following: rapeseed sclerotinia stem rot, rapeseed black shank, pepper blight, gray mold, tobacco blight, citrus blue mold, citrus green mold, fruit anthracnose, tomato early blight, wheat scab, corn ear rot, lychee downy mildew, pear black spot, or tea leaf blight.

[0017] In some embodiments, the plant disease is a combination of rapeseed sclerotinia stem rot and rapeseed black shank disease, or a combination of pepper blight and gray mold.

[0018] In some embodiments, the application involves diluting the liquid fermentation broth described in the second aspect and applying it to plants, plant seeds, plant fruits, or plant growth substrates by spraying, root irrigation, or fruit soaking.

[0019] Fourthly, this application provides a method for preventing and controlling plant diseases, which includes the following steps: applying the Streptomyces FLWZ-0001 described in the first aspect or the liquid fermentation broth described in the second aspect to plants, plant seeds, plant fruits or plant growth substrates.

[0020] Fifthly, this application provides the use of Streptomyces flavonoids FLWZ-0001 as described in the first aspect in screening active metabolites for the prevention and control of plant diseases.

[0021] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0022] 1. The *Streptomyces griseus* FLWZ-0001 strain provided in this application possesses broad-spectrum and highly effective antifungal activity, capable of simultaneously inhibiting multiple plant pathogens belonging to the fungi and oomycetes phyla. Oomycetes differ fundamentally from fungi in cell wall composition and metabolic pathways, making disease control more difficult. In vitro antifungal tests showed that this strain achieved inhibition rates exceeding 50% against all 13 tested pathogens. Specifically, it achieved inhibition rates of 80.37% and 84.7% against *Sclerotinia sclerotiorum* and *Penicillium fingernail*, respectively; furthermore, it achieved inhibition rates of 80.83% and 80.49% against *Phytophthora capsici* and *Phytophthora nicotineum*, respectively, belonging to the oomycetes phylum.

[0023] 2. The *Streptomyces griseus* FLWZ-0001 provided in this application exhibits excellent in vivo control efficacy against major diseases of various crops, especially demonstrating synergistic control capabilities against multiple diseases of the same crop. Pot experiments showed that a 10-fold diluted fermentation broth achieved a relative control efficacy of 87.39% against rapeseed sclerotinia stem rot and a relative control efficacy of up to 100% against rapeseed black shank, achieving simultaneous control of these two major rapeseed diseases. Fruit and pot experiments showed that a 10-fold diluted fermentation broth achieved a control efficacy of 98.15% against tomato gray mold and 77.62% against pepper blight. This ability to simultaneously and efficiently control multiple diseases on the same crop is significantly superior to existing biocontrol strains with narrow control spectra.

[0024] 3. The *Streptomyces griseus* FLWZ-0001 strain provided in this application can significantly inhibit the germination of pathogenic fungal spores, and its metabolites are clearly identified. Spore germination tests showed (see Example 6) that a 20-fold diluted fermentation supernatant inhibited the germination of *Botrytis cinerea* conidia by 89.62%. UPLC-MS analysis showed (see Example 4) that the main metabolite of this strain is a polyene compound (Philipmin), providing a material basis for its development as a biopesticide.

[0025] 4. The preparation method of the fermentation broth of Streptomyces flavonoids FLWZ-0001 provided in this application is simple, with diverse application methods, and is easy for industrial production. This strain can be applied by spraying, root irrigation, fruit soaking, etc., and is suitable for disease control in various crops such as rapeseed, pepper, citrus, and tomato, showing broad application prospects. Attached Figure Description

[0026] Figure 1 Plate contrast diagram showing the inhibition of *Streptomyces griseus* FLWZ-0001 by *Sclerotinia sclerotiorum* in rapeseed.

[0027] Figure 2 A phylogenetic tree for Streptomyces flavonoides FLWZ-0001 constructed based on the 16S rDNA sequence.

[0028] Figure 3This image shows the colony morphology of *Streptomyces griseus* FLWZ-0001 after 7 days of culture on an ISP-2 plate.

[0029] Figure 4 The UPLC-MS total ion chromatogram of the ethyl acetate extract of the fermentation broth of Streptomyces FLWZ-0001 shows that the metabolites mainly eluted at 4-6 min.

[0030] Figure 5 The UV absorption spectra of five major chromatographic peaks in the metabolites of Streptomyces flavonoids FLWZ-0001 are shown in sub-plots A, B, C, D, and E, which show characteristic absorption peaks at 322 nm, 337 nm, and 358 nm, respectively.

[0031] Figure 6 The mass spectra of five major chromatographic peaks in the metabolites of *Streptomyces flavonoids* FLWZ-0001 are shown, including sub-spectrums A, B, C, D, and E, corresponding to... Figure 5 The mass spectra of the compounds represented by each sub-plot. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0034] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before providing a detailed description of this application, the following terms and definitions are provided to better understand it.

[0036] 1. Preservation of the strain: The “Streptomyces murinus FLWZ-0001” mentioned in this application refers to the Streptomyces murinus strain deposited on September 5, 2024, at the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, Hubei Province, China), with accession number CCTCC NO: M20241923. Any implementation involving this strain refers to the use of biological material with the characteristics of this accession number.

[0037] 2. Fermentation broth: The "fermentation broth" mentioned in this application refers to the mixture of cultures obtained after inoculating *Streptomyces griseus* FLWZ-0001 into a suitable culture medium and culturing it in liquid for a certain period of time. It contains bacterial cells, unconsumed culture medium components, and secondary metabolites produced by bacterial metabolism. In specific embodiments of this application, the fermentation broth can be used directly or after dilution, filtration, or extraction.

[0038] 3. Plant Diseases: The term "plant diseases" in this application, unless otherwise specified, refers broadly to various diseases caused by pathogenic microorganisms infecting plants (including crops, cash crops, fruit trees, vegetables, etc.) that lead to stunted plant growth and development, reduced yield, or deteriorated quality. Preferred plant diseases in this application include, but are not limited to, rapeseed sclerotinia rot, rapeseed black shank, pepper blight, gray mold, tobacco blight, and postharvest diseases of citrus (such as blue mold and green mold).

[0039] 4. Fungal Pathogens: The "fungal pathogens" mentioned in this application refer to microorganisms belonging to the kingdom Fungi in biological taxonomy that can infect plants and cause diseases. Representative fungal pathogens in this application include, but are not limited to, *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Leptosphaeria maculans*, *Penicillium digitatum*, *Penicillium italicum*, *Colletotrichum gloeosporioides*, *Alternaria solani*, *Fusarium graminearum*, *Fusarium verticillioides*, *Peronophythora litchii*, *Alternaria gaisen*, and *Pestalotiopsis theae*.

[0040] 5. Oomycete Pathogens: The "oomycete pathogens" described in this application refer to microorganisms belonging to the phylum Oomycota within the kingdom Chromista in biological taxonomy, capable of infecting plants and causing diseases. Oomycetes differ significantly from true fungi in hyphal structure, cell wall composition (mainly containing cellulose, not chitin), reproductive methods, and sensitivity to fungicides. In this application, representative oomycete pathogens include, but are not limited to, *Phytophthora capsici* and *Phytophthora nicotianae*.

[0041] Example 1: Isolation and Identification of Streptomyces griseus FLWZ-0001

[0042] 1.1 Isolation of strains

[0043] The *Streptomyces flavovirens* FLWZ-0001 described in this application was isolated from the rhizosphere soil of healthy rapeseed plants from a severely diseased rapeseed field in Jiangxia District, Wuhan City, Hubei Province. Roots of healthy rapeseed plants were collected, and after shaking off the loose soil, root segments with attached rhizosphere soil were cut into 1-2 cm segments and placed in sterile water with shaking for 30 min to obtain a soil suspension. The soil suspension was spread onto Gao's No. 1 agar medium using a serial dilution method and incubated at 28 ℃ for 7-14 days. Single colonies were selected based on colony morphology, color, and size, purified, and preserved. Using *Sclerotinia sclerotiorum*, the pathogen of rapeseed sclerotinia rot, as a target, strains with antibacterial activity were screened using the plate confrontation method, resulting in a strain with significant antagonistic effect, designated FLWZ-0001.

[0044] Figure 1 This application demonstrates the plate confrontation inhibitory effect of *Streptomyces griseus* FLWZ-0001 on *Sclerotinia sclerotiorum* of rapeseed. Figure 1 It is evident that the mycelial growth of Sclerotinia sclerotiorum near the area marked by FLWZ-0001 was significantly inhibited, forming a clear inhibition zone, indicating that FLWZ-0001 has strong antagonistic activity against Sclerotinia sclerotiorum.

[0045] 1.2 Identification of strains

[0046] Total genomic DNA was extracted from strain FLWZ-0001 and amplified by PCR using universal primers for bacterial 16S rDNA. The universal primers are as follows:

[0047] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);

[0048] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0049] After purification, the PCR product was sent to a sequencing company (Shanghai Sangon Biotech) for sequencing. The obtained 16S rDNA sequence is as follows:

[0050] 16S rDNA of Streptomyces flavonoids FLWZ-0001 (SEQ ID NO.3):

[0051]

[0052] The sequence has been submitted to the NCBI public database (accession number: px964339). BLAST homology analysis was performed between this sequence and known sequences in the GenBank database. A phylogenetic tree was constructed using MEGA 11.0 software via the neighbor-joining method (e.g., Figure 2 As shown). Simultaneously, considering the strain's culture characteristics on ISP-2 medium (aerial hyphae are powdery and white, while substrate hyphae are yellowish-brown, with no soluble pigment production, such as...), Figure 3 Based on the results shown in the figure and the physiological and biochemical characteristics, the taxonomic position of strain FLWZ-0001 was determined to be Streptomyces murinus.

[0053] 1.3 Preservation of strains

[0054] The strain FLWZ-0001, identified as Streptomyces murinus, was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei Province, China) on September 5, 2024, and classified as Streptomyces murinus FLWZ-0001, with accession number CCTCC NO:M20241923.

[0055] Example 2: Preparation of fermentation broth of Streptomyces flavonoids FLWZ-0001

[0056] 2.1 Seed liquid preparation

[0057] Streptomyces griseus FLWZ-0001 preserved on slant culture was activated by streaking on ISP-2 plates and incubated at 28 ℃ for 7 days. Colony discs were then punched from the activated plates using a 4 mm diameter sterile punch and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of seed culture medium, with 5 colony discs inoculated into each flask. The flasks were incubated at 28 ℃ with shaking at 150 r / min for 4 days to obtain the seed culture.

[0058] The seed culture medium formula is as follows: mannitol 10 g / L, glucose 10 g / L, soybean peptone 10 g / L, calcium carbonate 0.5 g / L, dipotassium hydrogen phosphate 0.35 g / L, and the remainder is distilled water at natural pH.

[0059] 2.2 Liquid fermentation

[0060] The seed culture prepared in step 2.1 was inoculated at an inoculation rate of 10% (v / v) into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium. The flask was incubated at 28 ℃ with shaking at 150 r / min for 7 days to obtain the fermentation broth. Plate counts showed a viable cell concentration of 2 × 10⁻⁶ cells / mL. 8CFU / mL. This fermentation broth was used as the stock solution in subsequent examples.

[0061] The fermentation medium formula is as follows: corn flour 30 g / L, peanut meal 30 g / L, yeast extract 2.5 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 0.5 g / L, and the remainder is distilled water at natural pH.

[0062] Example 3: Determination of in vitro antibacterial activity of Streptomyces griseus FLWZ-0001

[0063] 3.1 Test pathogens

[0064] The plant pathogenic fungi used in this embodiment, along with their Latin names and corresponding crop diseases, are shown in Table 1. All pathogens were isolated, identified, and preserved in our laboratory.

[0065] Table 1. Determination of the antagonistic effect of FLWZ-0001 strain against the tested plant pathogenic fungi.

[0066]

[0067] Note: Data are expressed as mean ± standard deviation, and the same applies below.

[0068] 3.2 Confrontation Cultivation Method

[0069] In vitro antibacterial assays were performed using the plate confrontation culture method. Using a sterile punch (4 mm diameter), mycelial cakes were collected from the edge of activated pathogen colonies and inoculated into the center of PDA plates. *Streptomyces griseus* FLWZ-0001 was streaked symmetrically on both sides of the central mycelial cake using a sterile inoculating loop. Plates inoculated only with the pathogen and not with FLWZ-0001 served as blank controls. Each treatment was performed in triplicate. The plates were incubated at 28 °C. After the control group plates had fully colonized (approximately 5-7 days), the colony diameters of the pathogens in each treatment group and the control group were measured using the cross-sectional method. The inhibition rate was calculated using the following formula:

[0070] Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0071] 3.3 Experimental Results

[0072] The experimental results are shown in Table 1 above. The results indicate that *Streptomyces griseus* FLWZ-0001 exhibited varying degrees of antagonistic activity against all 13 tested pathogens, with inhibition rates exceeding 50%. Specifically, the inhibition rates against *Sclerotinias clerotiorum*, *Penicillium digitatum*, and *Penicillium italicum* (all fungi) reached 80.37%, 84.7%, and 84.45%, respectively. Particularly noteworthy is the high inhibition rate against *Phytophthora capsici* and *Phytophthora nicotianae* (both oomycetes), reaching 80.83% and 80.49%, respectively. In addition, this strain also has good inhibitory effects on Colletotrichum gloeosporioides, Alternaria solani, and Peronophythoralitchii, with an inhibition rate between 70% and 80%.

[0073] Example 4: Analysis of Metabolites from the Fermentation Broth of Streptomyces griseus FLWZ-0001

[0074] 4.1 Sample Preparation

[0075] The fermentation broth prepared in Example 2 was centrifuged at 4 °C and 10,000 rpm for 10 min, and the supernatant was collected. The supernatant was mixed with an equal volume of ethyl acetate, and the mixture was thoroughly extracted by shaking in a separatory funnel. After standing and separating the layers, the ethyl acetate phase was collected. The extraction was repeated three times, and the ethyl acetate phases were combined. The ethyl acetate phase was evaporated to dryness under reduced pressure at 40 °C. The residue was dissolved in a small amount of chromatographic grade methanol and filtered through a 0.22 μm organic phase filter membrane (Millipore, catalog number: SLGPR33RB) to obtain the sample to be tested.

[0076] 4.2 UPLC-MS Analysis

[0077] Samples were analyzed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). Instrument model: Waters XEVO TQD UPLC / MS system (Waters Corporation, USA); Column: Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm); Column temperature: 40 ℃; Flow rate: 0.45 mL / min; Injection volume: 2 μL; Mobile phase: A = water + 0.2% acetic acid, B = acetonitrile + 0.2% acetic acid; Gradient elution program: 0–0.2 min, 95% A; 0.2–8.2 min, 95% A → 100% B; 8.2–9.7 min, 100% B; 9.7–11.30 min, 100% B → 95% A; 11.30–13.00 min, 95% A.

[0078] Mass spectrometry conditions: ion source: ESI+; capillary voltage: 3.5 kV; desolvation temperature: 450 ℃; desolvation nitrogen flow rate: 1000 L / h; cone voltage: 30 V; collision energy: 12 V.

[0079] 4.3 Experimental Results

[0080] UPLC-MS analysis results are as follows Figure 4 As shown, the metabolites of strain FLWZ-0001 mainly eluted within 4-6 min, presenting a set of chromatographic peaks. The retention times of the five main chromatographic peaks were approximately 4.36 min, 4.49 min, 4.72 min, 5.22 min, and 5.52 min, respectively.

[0081] The compounds corresponding to these five main chromatographic peaks were analyzed by ultraviolet absorption spectroscopy, and the results are as follows: Figure 5 As shown. Figure 5 It includes sub-graphs A, B, C, D, and E, which correspond to... Figure 4 The UV absorption spectra of five main chromatographic peaks with retention times of approximately 4.36 min, 4.49 min, 4.72 min, 5.22 min, and 5.52 min were obtained. Figure 5 As can be seen, the compounds corresponding to these five chromatographic peaks all exhibit characteristic triple absorption at 322 nm, 337 nm, and 358 nm, which are typical ultraviolet absorption characteristics of polyene compounds, indicating that these compounds are all polyene homologues.

[0082] Further mass spectrometry analysis was performed on the compounds corresponding to these five main chromatographic peaks, and the results are as follows: Figure 6 As shown. Figure 6 It includes sub-graphs A, B, C, D, and E, which correspond to... Figure 5The mass spectra of the compounds represented by A, B, C, D, and E in the diagram are shown. Combining the results of ultraviolet absorption spectroscopy and mass spectrometry analysis, the main component of the strain's metabolite can be identified as filipins, belonging to the polyene macrolide antibiotic class.

[0083] Example 5: Pot experiment on the control of sclerotinia stem rot and black shank disease in rapeseed using Streptomyces griseus FLWZ-0001.

[0084] 5.1 Test Materials

[0085] The rapeseed variety tested was Zhongshuang 11 (purchased from an agricultural supply store, produced by Wuhan Zhongyou Seed Industry Technology Co., Ltd.).

[0086] Test pathogens: Sclerotinia sclerotiorum and Leptosphaeria maculans (isolated, identified and preserved in our laboratory).

[0087] Test fermentation broth: The original FLWZ-0001 fermentation broth prepared according to the method in Example 2, diluted with sterile water to the required ratio before use.

[0088] 5.2 Experiment on the control of sclerotinia stem rot by detached rapeseed leaves

[0089] Take the third true leaf from a rapeseed plant cultured for 30 days, rinse it thoroughly with tap water, then surface disinfect it in a 2% sodium hypochlorite solution for 2 minutes, rinse it three times with sterile water, and air dry it. Place the leaf in a petri dish with moistened filter paper at the bottom. Using a spray method, spray different dilutions of the fermentation broth (10x, 20x, 50x, 100x) evenly onto the leaf surface, with sterile water spraying serving as a control. After the leaf surface has dried, inoculate the center of each leaf with a 5 mm diameter Sclerotinia sclerotiorum mycelium cake. Each treatment is repeated 5 times. Place the petri dishes in a culture room at 20 ℃ with 16 h light / 8 h darkness. After 2 days, measure the diameter of the lesions and calculate the relative control efficacy using the following formula:

[0090] Relative efficacy (%) = (Lesion area in control group - Lesion area in treatment group) / Lesion area in control group × 100%

[0091] The experimental results are shown in Table 2.

[0092] Table 2. Effects of FLWZ-0001 fermentation broth (leaf method) on the control of sclerotinia stem rot in rapeseed.

[0093]

[0094] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05), and the same applies below.

[0095] As shown in Table 2, the 10-fold dilution of fermentation broth achieved a control efficacy of 80.11% against detached leaves of rapeseed sclerotinia stem rot, which was significantly higher than other dilution ratios.

[0096] 5.3 Experiment on the control of sclerotinia stem disease in potted rapeseed

[0097] Rapeseed seedlings were cultivated to the 3-4 leaf stage, and healthy plants with uniform growth were selected. Different dilutions of the fermentation broth (5x, 10x, 20x, 50x, and 100x) were evenly sprayed onto the surface of the rapeseed stems using a spraying method, with sterile water serving as a control. Forty-eight hours after treatment, 5mm diameter *Sclerotium sclerotiorum* mycelium blocks were placed on the stems, ensuring the mycelial surface was in close contact with the stem, and then fixed with sealing film. Each treatment was replicated 10 times. Forty-eight hours after inoculation, the length of stem lesions was observed and measured, and the relative control efficacy was calculated using the following formula:

[0098] Relative efficacy (%) = (Leg length of control group - lesion length of treatment group) / lesion length of control group × 100%.

[0099] The experimental results are shown in Table 3.

[0100] Table 3. Effects of FLWZ-0001 fermentation broth on the control of sclerotinia stem rot in rapeseed in pot experiments.

[0101]

[0102] As shown in Table 3, the stem lesion length treated with 5-fold and 10-fold fermentation broth dilutions was significantly shorter than that of the control, with relative control efficacy reaching 94.47% and 87.39%, respectively.

[0103] 5.4 Experiment on root control of blackleg disease in potted rapeseed

[0104] Rapeseed seedlings were cultivated to the 3-4 leaf stage, and healthy plants with uniform growth were selected. Using the root drenching method, 20 mL of fermentation broth diluted at different ratios (10x, 20x, 50x, and 100x) was applied to each pot, with sterile water used as a control. After 48 h of treatment, the plants were inoculated with a suspension of black shank spores (1×10⁻⁶) using the root drenching method. 6 CFU / mL), 10 mL per pot. Each treatment had 10 replicates. 14 days post-inoculation, the disease incidence in rapeseed seedlings was assessed and graded according to a 0-5 scale:

[0105] Grade 0: No diseased tissue was observed in the cross-section of the plant;

[0106] Grade 1: The infected area on the cross-section of the plant is < 25%;

[0107] Level 2: The infected area accounts for 25%-50% of the cross-sectional area;

[0108] Level 3: Infected area accounts for 50%-75% of the cross-sectional area;

[0109] Grade 4: Infected area > 75%, with little or no shrinkage of the infected tissue;

[0110] Level 5: 100% infected area, infected tissues shrivel, and plant dies.

[0111] The disease index and prevention and control effectiveness are calculated using the following formulas:

[0112] Disease index = ∑ (number of diseased plants at each level × level) / (total number of plants surveyed × 5) × 100;

[0113] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0114] The experimental results are shown in Table 4.

[0115] Table 4. Effects of FLWZ-0001 fermentation broth on the control of blackleg disease in rapeseed in pot experiments.

[0116]

[0117] As shown in Table 4, the 10-fold dilution of the fermentation broth achieved 100% control efficacy against black shank disease in potted rapeseed, while the 20-fold dilution achieved 77.09% control efficacy, which was significantly higher than the control.

[0118] Example 6: Application of Streptomyces boulardii FLWZ-0001 in the control of gray mold.

[0119] 6.1 Inhibition test on Botrytis cinerea spore germination

[0120] The fermentation broth prepared in Example 2 was centrifuged at 4 °C and 10,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm bacterial filter (Millipore, catalog number: SLGPR33RB) to obtain the fermentation supernatant filtrate. The fermentation supernatant filtrate was mixed with melted PDA medium (approximately 45 °C) to a supernatant concentration of 20%, and immediately poured into petri dishes to prepare plates. 0.1 mL of a Botrytis cinerea spore suspension (1×10⁻⁶) was then added. 6 Spores / mL were evenly spread on agar plates. An equal volume of sterile water was used as a blank control instead of the fermentation supernatant. Each treatment was repeated three times. The plates were incubated at 20°C, and spore germination was examined under a microscope at 2 h, 3.5 h, and 5 h to calculate the spore germination rate. The spore germination rate and inhibition rate were calculated using the following formulas:

[0121] Spore germination rate (%) = (Number of germinating spores / Total number of observed spores) × 100%;

[0122] Inhibition rate (%) = (Control germination rate - Treatment germination rate) / Control germination rate × 100%.

[0123] The experimental results are shown in Table 5.

[0124] Table 5. Effects of fermentation supernatant filtrate of strain FLWZ-0001 on the germination of conidia of Botrytis cinerea.

[0125]

[0126] As shown in Table 5, after 5 hours of culture, the 20-fold dilution of fermentation supernatant inhibited the germination of Botrytis cinerea conidia by 89.62%, significantly suppressing spore germination.

[0127] 6.2 Experiment on control of gray mold in tomato fruit

[0128] Select mature tomato fruits of uniform size and free from pests and diseases. Disinfect the surface by soaking in a 2% sodium hypochlorite solution for 2 minutes, rinse three times with sterile distilled water, and air dry. Make a cross-shaped incision (approximately 3 mm long and 2 mm deep) on the equator of the fruit using a sterile scalpel. Mix the *Botrytis cinerea* spore suspension with fermentation broth diluted at different ratios (10x, 20x, 30x, 50x, and 100x) to achieve a final spore concentration of 1×10⁻⁶. 5 Spores / mL. 10 μL of the mixture was added dropwise to the tomato wound. Sterile water was used as a control instead of the fermentation broth. Each treatment was repeated 10 times. The fruits were cultured in a 20 ℃, 16 h light / 8 h dark environment. Disease incidence was assessed after 2 days, and the disease was graded according to a 0-4 scale.

[0129] Grade 0: No disease spots on the fruit surface;

[0130] Grade 1: The fruit surface has a few disease spots;

[0131] Grade 2: The area of ​​lesions accounts for less than 20% of the fruit surface area;

[0132] Grade 3: Lesions cover 20%-50% of the fruit surface area;

[0133] Grade 4: The area of ​​lesions accounts for more than 50% of the fruit surface area.

[0134] The disease index and prevention and control effectiveness are calculated using the following formulas:

[0135] Disease index = ∑ (number of fruits at each level × level) / (total number of fruits surveyed × 4) × 100;

[0136] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0137] The experimental results are shown in Table 6.

[0138] Table 6. Effects of FLWZ-0001 fermentation broth on the control of gray mold in grapes (experimental study on tomato fruit).

[0139]

[0140] As shown in Table 6, the 10-fold and 20-fold dilutions of the fermentation broth achieved fruit control efficacy of 98.15% and 90.63% against tomato gray mold, respectively, which were significantly higher than other treatments.

[0141] Example 7: Application of Streptomyces griseus FLWZ-0001 in the control of Phytophthora blight in peppers

[0142] 7.1 Test Materials

[0143] Chili pepper variety tested: Zaozha 2 (purchased from an agricultural supply store, manufacturer: Jiangxi Nanchang Fengyu Seed Industry Co., Ltd.). Pathogen tested: *Phytophthora capsici* (isolated, identified, and preserved in our laboratory). Fermentation broth tested: FLWZ-0001 fermentation broth stock solution prepared according to the method in Example 2, diluted with sterile water before use.

[0144] Required multiplier.

[0145] 7.2 Test Methods

[0146] Chili seedlings were cultivated to the 3-4 leaf stage, and healthy plants with uniform growth were selected. Using the root drenching method, 20 mL of fermentation broth diluted at different ratios (10x, 20x, 50x, and 100x) was applied to each pot, allowing the bacterial solution to fully penetrate the substrate around the roots. Sterile water was used as a control. After 24 hours of treatment, a suspension of *Phytophthora capsici* spores (1×10⁻⁶) was inoculated using the root drenching method. 6 CFU / mL), 10 mL per pot. Eight replicates were set up for each treatment. Fourteen days post-inoculation, the disease incidence in pepper seedlings was assessed and graded according to a 0-5 scale:

[0147] Grade 0: The entire plant is disease-free;

[0148] Grade 1: Less than 20% of lateral branches are affected, or water-soaked lesions appear at the base of the stem;

[0149] Grade 2: 20%-50% of lateral branches are affected, or rotten spots appear at the base of the stem, but the plant does not wilt;

[0150] Level 3: 50%-75% of lateral branches are affected, or rotten spots appear at the base of the stem, and the plant shows signs of reversible wilting.

[0151] Level 4: More than 75% of lateral branches are infected, some branches die, or the plant shows irreversible wilting.

[0152] Level 5: The entire plant has died.

[0153] Disease index and prevention and control effectiveness are calculated using the following formula:

[0154] Disease index = ∑ (number of diseased plants at each level × level) / (total number of plants surveyed × 5) × 100;

[0155] Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0156] 7.3 Experimental Results

[0157] The experimental results are shown in Table 7.

[0158] Table 7. Effects of FLWZ-0001 fermentation broth on the control of Phytophthora capsici in pot experiments.

[0159]

[0160] As shown in Table 7, the 10-fold and 20-fold dilutions of the fermentation broth achieved control efficacy of 77.62% and 70.53% against Phytophthora blight in potted plants, respectively, significantly higher than other dilutions. These results indicate that *Streptomyces griseus* FLWZ-0001 has a good control effect against Phytophthora blight in peppers.

[0161] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A strain of Streptomyces murinus, which was deposited at the China Center for Type Culture Collection on September 5, 2024, with the following classification and name: Streptomyces murinus FLWZ-0001; accession number: CCTCC NO: M20241923; location: Wuhan University, Wuhan, China.

2. The liquid fermentation broth of *Streptomyces flavonoids* FLWZ-0001 as described in claim 1 is prepared by a method comprising the following steps: (1) Seed culture: FLWZ-0001 of Streptomyces gravidarum was inoculated into seed culture medium and cultured at 28 °C and 150 r / min for 4 days to obtain seed culture; (2) Liquid fermentation: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 10%, and culture at 28 °C and 150 r / min for 7 days to obtain the fermentation liquid.

3. The use of the Streptomyces flavonoids FLWZ-0001 of claim 1 or the liquid fermentation broth of claim 2 in the preparation of microbial agents for the prevention and control of plant diseases.

4. The application according to claim 3, wherein the plant disease is a disease caused by fungal pathogens and / or a disease caused by oomycete pathogens.

5. The application according to claim 4, wherein the fungal pathogens include at least one of *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Blacklegs*, *Penicillium finger*, *Penicillium italicum*, *Colletotrichum gloeosporioides*, *Alternaria alternata*, *Fusarium graminearum*, *Fusarium verticillatum*, *Phytophthora licheniformis*, *Alternaria peariformis*, and *Phytophthora chamaeja*; and the oomycete pathogens include at least one of *Phytophthora capsici* and *Phytophthora tumefaciens*.

6. The application according to claim 3, wherein the plant disease is at least one of the following: rapeseed sclerotinia stem rot, rapeseed black shank, pepper blight, gray mold, tobacco blight, citrus blue mold, citrus green mold, fruit anthracnose, tomato early blight, wheat scab, corn ear rot, lychee downy mildew, pear black spot, or tea leaf blight.

7. The application according to claim 6, wherein the plant disease is a combination of rapeseed sclerotinia stem rot and rapeseed black shank, or a combination of pepper blight and gray mold.

8. The application according to claim 3, wherein the application is to apply the diluted liquid fermentation liquid of claim 2 to plants, plant seeds, plant fruits or plant growth substrates by spraying, root irrigation or fruit soaking.

9. A method for preventing and controlling plant diseases, comprising the following steps: Apply the Streptomyces flavonoids FLWZ-0001 of claim 1 or the liquid fermentation broth of claim 2 to plants, plant seeds, plant fruits or plant growth substrates.

10. The use of the Streptomyces flavonoides FLWZ-0001 of claim 1 in screening for active metabolites for the prevention and control of plant diseases.

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