A strain of brown Streptomyces GXMU-J15 and its application

Abstract

This invention relates to the field of microbial technology, and more particularly to a strain of *Streptomyces brownii* GXMU-J15 and its applications. *Streptomyces brownii* GXMU-J15 of this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 20, 2023, with accession number GDMCC No:63901. *Streptomyces brownii* GXMU-J15 exhibits significant broad-spectrum antibacterial activity against various plant pathogens, achieving an inhibition rate of 78.99% against *Phytophthora tobaccoii*. It can increase the plant height, stem diameter, and both above-ground and underground fresh and dry weight of tobacco plants, and inhibit the mycelial growth of *Phytophthora tobaccoii*, providing a basis for the prevention and control of tobacco black shank disease.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a strain of Streptomyces brownii GXMU-J15 and its applications. Background Technology

[0002] Tobacco black shank can infect tobacco plants at any stage of growth. It infects the plant through the rapid spread of its zoospores and the formation of appressoriums at the tips of germination tubes, leading to root rot, necrosis of stems and leaves, and ultimately plant death. Tobacco black shank is one of the most widespread and destructive soil-borne diseases in tobacco cultivation. Its pathogen is *Phytophthora nicotineae*, and this disease occurs in all major tobacco-growing areas, causing significant economic losses and posing a serious challenge to sustainable development.

[0003] Currently, tobacco black shank is difficult to control, and the main management measures include breeding resistant varieties, crop rotation, and the use of chemical fungicides. The selection cycle for resistant varieties is long, and their resistance is determined by genes. While resistance conferred by multiple genes is more durable, it gradually weakens over time, and pathogen populations can quickly adapt to the selection pressure brought by resistant genes. In agricultural production, intensive continuous cropping is common, but tobacco is sensitive to and unsuited to this model, leading to a decline in yield and quality. Furthermore, the effects of autotoxicity disrupt the soil micro-ecosystem and reduce soil fertility, further contributing to the outbreak of soil-borne diseases. Chemical fungicides are one of the important means of controlling this disease; however, the number of available fungicides is currently limited, and long-term use of fungicides can damage the health of the soil environment and easily lead to drug resistance in pathogens. Therefore, finding efficient, low-toxicity, and environmentally friendly solutions to prevent tobacco black shank caused by Phytophthora indicum is crucial. Summary of the Invention

[0004] The purpose of this invention is to provide a strain with broad-spectrum antibacterial activity, good biocontrol effect, and wide application range for the control of Phytophthora tobaccois. In particular, it relates to a strain of Streptomyces brownii GXMU-J15 and its applications.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a strain of *Streptomyces brownii* (… Streptomyces sp.) GXMU-J15 is deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 20, 2023, with accession number GDMCC No:63901.

[0006] The present invention also provides the aforementioned *Streptomyces brownii* ( StreptomycesApplication of sp.) GXMU-J15 in the preparation of products for preventing and controlling plant diseases caused by fungi.

[0007] Preferably, the fungus includes Phytophthora tobaccoii (Phytophthora indicum). Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunate ), Fusarium graminearum ( Fusarium pseudograsses ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. from Cuba One or more of the following.

[0008] Preferably, the product includes any one of fermentation broth, sterile supernatant, and crude fermentation extract.

[0009] This invention also provides a brown Streptomyces ( Streptomyces The fermentation broth of GXMU-J15, sp., is composed of the aforementioned Streptomyces brownii (sp.) Streptomyces It was prepared by sp.) GXMU-J15.

[0010] This invention also provides a brown Streptomyces ( Streptomyces The preparation method of fermentation broth of sp. GXMU-J15 includes the following steps: The brown Streptomyces ( Streptomyces GXMU-J15 sp. was inoculated into the culture medium and cultured at 25-37℃ for 1-7 days to obtain the fermentation broth. The rotation speed during cultivation is 150~250 rpm; The culture medium is ISP2 liquid medium.

[0011] The present invention also provides the application of the fermentation liquid or the fermentation liquid prepared by the preparation method described above in the prevention and control of plant diseases caused by fungi and the improvement of plant growth performance; The fungi include Phytophthora nicotineae ( Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunate ), Fusarium graminearum ( Fusarium pseudograsses ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. from Cuba One or more of the following; The plant in question is tobacco.

[0012] This invention also provides a brown Streptomyces ( Streptomyces The sterile supernatant of sp.) GXMU-J15, wherein the method for preparing the sterile supernatant is as follows: Centrifuge the fermentation broth for 8-12 minutes, collect the supernatant, filter the supernatant, collect the filtrate, and obtain a sterile supernatant. The centrifuge speed is 10000~14000 rpm; The filtration is membrane filtration, and the pore size of the membrane is 0.2~0.24μm.

[0013] The present invention also provides the application of the sterile supernatant in the prevention and control of plant diseases caused by fungi and the improvement of plant growth performance; The fungi include Phytophthora nicotineae ( Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunate ), Fusarium graminearum ( Fusarium pseudograsses ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. from Cuba One or more of the following; The plant in question is tobacco.

[0014] This invention also provides a brown Streptomyces fungus for preventing and controlling plant diseases caused by fungi ( Streptomyces The crude fermentation extract of sp. GXMU-J15, wherein the preparation method of the crude fermentation extract is as follows: The sterile supernatant was extracted with ethyl acetate 2-4 times, the extracts were combined, and concentrated to dryness under reduced pressure to obtain the crude fermentation extract. The volume ratio of acetic acid and ethyl ester in the ethyl acetate is 1~2:1~2; The temperature for vacuum concentration is 38~42℃.

[0015] The present invention has the following advantages: This invention isolates and screens a strain of *Streptomyces brownii* from tobacco rhizosphere soil. Streptomyces Streptomyces bruncii sp. GXMU-J15 exhibits significant broad-spectrum antifungal activity against various plant pathogens, demonstrating the ability to slow pathogen infection and reduce plant disease incidence. Its broad antibacterial spectrum is evident in its ability to inhibit Phytophthora tobaccoii (sp.) Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae), fruit mold ( Pythium aphanidermatum ), Curvularia crescentis ( Curvularia lunate ), Fusarium graminearum ( Fusarium pseudograsses ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. from Cuba ) growth.

[0016] The *Streptomyces brownii* GXMU-J15 strain exhibited a 78.99% inhibition rate against *Phytophthora tobaccoii*, with a 10% fermentation broth showing an antibacterial activity of 60.27%. It also increased tobacco plant height, stem diameter, and the fresh and dry weights of the aboveground and underground parts by 34.28%, 25.27%, 80.90%, 50.07%, 91.18%, and 37.5%, respectively. This was achieved at a pathogen concentration of 1×10⁻⁶. 7 The control effect of indoor potted plants at CFU / mL is over 65.92%; it can also cause the ends of Phytophthora tobacco hyphae to swell and branch, destroy the cell wall structure, thereby inhibiting the growth of Phytophthora tobacco hyphae, and can be applied to the control of tobacco black shank disease.

[0017] Preservation Instructions

[0018] Brown Streptomyces ( Streptomyces sp.)GXMU-J15 was deposited on October 20, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:63901. Attached Figure Description

[0019] Figure 1 The colony growth of strain GXMU-J15 in different culture media (A is ISP1 medium; B is ISP2 medium; C is ISP3 medium; D is ISP4 medium; E is ISP5 medium; F is ISP6 medium; G is ISP7 medium; H is Gao's No. 1 medium). Figure 2 Microscopic morphological characteristics of strain GXMU-J15 (A is the result of optical microscopy, B and C are the results of scanning electron microscopy). Figure 3 Physiological and biochemical characteristics of strain GXMU-J15 (A: L-arabinose; B: lactose; C: D-fructose; D: L-alanine; E: L-lysine; F: L-serine; G: yeast extract; H: L-tryptophan; I: tryptone; J: L-threonine; K: L-valine; L: milk coagulation and peptone test; M: gelatin test; N: Volta-Pyle test). Figure 4 Phylogenetic tree of 16S rDNA for strain GXMU-J15; Figure 5 A phylogenetic tree of the whole genome of strain GXMU-J15; Figure 6 Results of the plate confrontation method (A and C are control groups; B and D are treatment groups); Figure 7 The inhibitory effect of strain GXMU-J15 on plant pathogens; Figure 8 The inhibitory effect of strain GXMU-J15 on Phytophthora indicum (A: Inhibitory effect of fermentation broth of strain GXMU-J15 on Phytophthora indicum in different culture media, where (a) control, (b) ISP2 fermentation broth, (c) PDB fermentation broth, (d) LB fermentation broth; B: Poison plate containing 10% ISP2 fermentation broth of strain GXMU-J15, where (a) control, (b) treatment; C: Inhibitory effect of gas produced by strain GXMU-J15 on Phytophthora indicum, where (a) control, (b) treatment; D: Inhibitory effect of modified ISP2 fermentation broth of strain GXMU-J15 on Phytophthora indicum, where (a) control, (b) treatment; E: Inhibitory effect of crude extract of strain GXMU-J15 dissolved in 100% methanol on Phytophthora indicum, where (a) control, (b) treatment). Figure 9 The growth-promoting effect of strain GXMU-J15 (A is protease, B is amylase, C is chitinase, D is β-glucanase, E is cellulase, F is ACC deaminase, G is HCN, H is siderophore, I is nitrogen fixation, J is organic phosphorus, K is inorganic phosphorus, L is IAA, "+" indicates positive, "-" indicates negative). Figure 10 The pot growth-promoting effect of strain GXMU-J15 (A is the control, B is the treatment). Figure 11 The effects of different treatments on tobacco growth (A represents growth at 7 days, B represents growth at 14 days, and C represents growth at 21 days). Detailed Implementation

[0020] In this invention, *Streptomyces brownii* ( Streptomyces The viable cell concentration of the fermentation broth of sp. GXMU-J15 was 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL.

[0021] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0022] The tobacco phytophyte described in the embodiments of the present invention ( Phytophthora nicotianae ) and basal root bead mold ( Berkeleyomyces basicola (Provided by Guangxi Tianyuan Biochemical Co., Ltd.)

[0023] The Staphylococcus aureus described in the embodiments of the present invention ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunate ), fruit mold ( Pythium aphaniderm ), Fusarium graminearum ( Fusarium pseudogramineum ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. cubense,Foc All materials were provided by the Key Laboratory of Polysaccharide Materials and Modification, College of Marine and Biotechnology, Guangxi University for Nationalities.

[0024] Example 1

[0025] strain isolation

[0026] Soil samples were collected from dry tidal flat sandy soil in Beihai, Guangxi (21.6243290°N, 108.9088834°E). The samples were placed in sterile self-sealing bags and stored in sterile plastic bags at -20°C. Actinomycetes were isolated and purified using the plate coating method.

[0027] The specific steps are as follows: Soil samples were prepared into concentrations of 10 using the dilution plating method. -3 10 -4 10 -5 Soil suspension was evenly spread using a sterile spreader, sealed and stored in a 28°C constant temperature incubator, and observed in a timely manner. After bacteria grew, it was transferred to a new solid culture medium plate and repeatedly operated on ISP2 solid culture medium by streak plating until a stable single colony of bacteria was obtained. It was then stored in a 20% glycerol solution to obtain strain GXMU-J15.

[0028] Example 2

[0029] Strain identification

[0030] (1) Morphological identification

[0031] The strain GXMU-J15 obtained in Example 1 was streaked and inoculated onto Gao's No. 1 and ISP 1-7 solid media, respectively, and cultured at 28°C for 3 days. The morphology of single colonies was observed and recorded.

[0032] Gao's No. 1 solid culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. All culture media were sterilized at 121℃ for 20 min after preparation. The following solid culture media were prepared: ISP1 (tryptone 5g / L, yeast extract 3g / L, agar 15g / L), ISP2 (maltose 10g / L, glucose 4g / L, yeast extract 4g / L, agar 20g / L), ISP3 (oat 20g / L, FeSO4·7H2O 0.001g / L, MnCl2·4H2O 0.001g / L, ZnSO4·7H2O 0.001g / L, agar 18g / L), and ISP4 (soluble starch 10g / L, K2HPO4 1g / L, MgSO4·7H2O 1g / L, NaCl 1g / L, (NH4)2SO4 2g / L, CaCO3). 2 g / L, FeSO4·7H2O 0.001 g / L, MnCl2·4H2O 0.001 g / L, ZnSO4·7H2O 0.001 g / L, agar 20 g / L), ISP5 solid medium (L-asparagine 1 g / L, K2HPO4 1 g / L, FeSO4·7H2O 0.001 g / L, MnCl2·4H2O 0.001 g / L, ZnSO4·7H2O 0.001 g / L, agar 20 g / L), ISP6 solid medium (tryptone 15 g / L, lycine peptone 5 g / L, yeast extract 1 g / L, ferric ammonium citrate 0.5 g / L, K2HPO4 1 g / L, Na2S2O3 0.08 g / L, agar 15 g / L), ISP7 solid medium (L-asparagine 1 g / L, L-tyrosine 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, Ho-Le trace elements 1 mL / L, glycerol 15 mL / L, agar 20 g / L) and Ho-Le trace elements (boric acid 2.85 g / L, MnCl2·4H2O 1.8 g / L, FeSO4·7H2O 1.36 g / L, sodium tartrate 1.77 g / L, CuCl2·2H2O 26.9 mg / L, ZnCl2 20.8 mg / L, CoCl2·6H2O 40.4 mg / L, Na2MoO4·2H2O) Prepare the culture medium (25.2 mg / L) yourself. After all the culture media are prepared, sterilize them at 115℃ for 20 min.

[0033] The colony morphology of strain GXMU-J15 is as follows Figure 1 As shown, it is round with an irregular, wrinkled surface. It is white in the early stages of growth and turns brown in the later stages. It has white hyphae at the edges, does not produce soluble pigments, and has an irregular colony shape.

[0034] Morphology under an optical microscope as follows Figure 2 As shown, strain GXMU-J15 has well-developed aerial hyphae, which are white cottony spores. The spore filaments are branched, forming a loose spiral shape. Some aerial hyphae are differentiated into straight, flexible, hook-shaped to loose or tight spiral spore filaments.

[0035] (2) Physiological and biochemical characteristics

[0036] The physiological and biochemical characteristics of strain GXMU-J15 were determined according to some of the methods recommended in "Systematic Classification Techniques of Actinomycetes". The results are shown in Table 1 and... Figure 3 As shown in Table 1, + indicates a positive reaction and indicates a negative reaction.

[0037] Table 1. Physiological and biochemical characteristics of strain GXMU-J15

[0038] The results of physiological and biochemical characteristics are shown in Table 1 and Figure 3 As shown, the physiological and biochemical characteristics test results indicate that strain GXMU-J15 can utilize lactose, sucrose, D-xylose, L-arabinose, D-maltose, and D-fructose as its sole carbon source, and L-lysine, L-alanine, L-tryptophan, L-threonine, L-serine, yeast extract, and peptone as its sole nitrogen source. The gelatin test, nitrate reduction test, milk peptone and coagulation test, and methyl red test are all positive.

[0039] (3) Molecular identification

[0040] DNA extraction of strain GXMU-J15 was performed using the commonly used Chelex-100 method.

[0041] After extracting DNA from strain GXMU-J15, a PCR reaction was performed.

[0042] PCR reaction system: 25 μL of 2×RapidTaqMasterMix, 1 μL each of Primer1 and Primer2, 2 μL of Template DNA, and ddH2O added to a final volume of 50 μL. Rapid Taq Master Mix was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] PCR reaction conditions: 95°C pre-denaturation for 5 min, 95°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 2 min, 40 cycles, and a final extension at 72°C for 10 min.

[0044] The primer sequence used is:

[0045] Primer1(27F):5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1); Primer2(1541R): 5'-AAGGAGGTGATCCAGCC-3' (SEQ ID NO. 2).

[0046] The amplified PCR stock solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing, and the 16S rDNA sequence of the strain (SEQ ID NO.3) was obtained. The obtained sequence was compared with sequences in the GenBank database using BLAST analysis, and a molecular identification cluster map was constructed. The results are as follows: Figure 4 As shown, Streptomyces GXMU-J15 and S. lusitanus CGMCC 4.1745 T , S. thermocarboxydus CGMCC 4.1883 T Gather together a branch.

[0047] Strain GXMU-J15 was cultured in ISP2 liquid medium at 28°C for 7 days. The bacterial cells of strain GXMU-J15 were collected by centrifugation, dehydrated using a freeze dryer (FD, Biocool), and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for whole-genome sequencing. The obtained sequences were uploaded to the Type (Strain) Genome Server (TYGS) of the German Microbiology and Cell Culture Collection (DMC) to construct a genome clustering map using default parameters. The results are as follows: Figure 5 As shown, strain GXMU-J15 is named Streptomyces sp. .

[0048] Based on morphological characteristics, physiological and biochemical characteristics, and molecular biological identification results, strain GXMU-J15 was identified as belonging to the genus Streptomyces. Streptomyces. Streptomyces sp. brownis Streptomyces fuscus .

[0049]

[0050] Example 3

[0051] (1) Flat standoff method

[0052] Strain GXMU-J15 was inoculated onto ISP2 medium and incubated at 28°C until single colonies appeared. A 7.5 mm *Phytophthora tobaccoeris* cake was placed in the center of a 90 mm PDA (potato starch 200 g / L, glucose 20 g / L, agar 20 g / L) plate. Strain GXMU-J15 was inoculated on both sides, 20 mm from the center of the PDA plate. The plates were incubated at 28°C for 5 days, serving as the treatment group. The mycelial growth of *Phytophthora tobaccoeris* at the opposing culture sites was observed under an optical microscope and a scanning electron microscope. The control group consisted of the uninoculated sections of strain GXMU-J15.

[0053] Microscopic observation revealed that strain GXMU-J15 significantly inhibited the mycelial growth of *Phytophthora tobaccoeris*. Figure 6 As shown, normally growing hyphae have a smooth and transparent surface. In contrast, the hyphae of *Phytophthora indicum* in the confrontation culture exhibit deformities, swelling, and multi-branching, and the hyphae lyse.

[0054] (2) Determination of the ability to antagonize plant pathogens

[0055] The strain GXMU-J15 was inoculated onto ISP2 medium and incubated at 28°C until a single colony grew.

[0056] Using a 7.5mm punch, a single colony of strain GXMU-J15 was picked up entirely and placed onto PDA solid medium inoculated with the pathogenic fungus, serving as the experimental group. A control group was also set up, inoculated only with the pathogenic fungus on PDA medium, without inoculating strain GXMU-J15. Both groups were incubated at 28℃. Once the control group had completely covered the entire plate, the growth of the pathogenic fungus in the experimental group was observed, recorded, and the inhibition rate was calculated.

[0057] The results are shown in Table 2 and Figure 7 As shown, strain GXMU-J15 exhibits antagonistic effects against a variety of plant pathogens, including Phytophthora tobaccoii, Leuconostoc medusa, Pythium spp., Staphylococcus aureus, Banana anthracnose, Curvularia crescentis, Fusarium pseudograssii, and Fusarium oxysporum, with an inhibition rate of up to 78.99% against Phytophthora tobaccoii.

[0058] Table 2. Inhibitory effect of strain GXMU-J15 on plant pathogens (n=3)

[0059] Example 4

[0060] Determination of the antibacterial activity of strain GXMU-J15 against Phytophthora nicotineae

[0061] (1) Preparation of fermentation broth of strain GXMU-J15: strain GXMU-J15 was inoculated into PDA, LB and ISP2 liquid culture media respectively, and cultured at 28℃ and 200rpm for 7 days.

[0062] (2) Preparation of sterile supernatant: The fermentation broth of strain GXMU-J15 was centrifuged at 12000 rpm for 10 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was taken to obtain sterile supernatant.

[0063] The plate confrontation method was used. A 7.5 mm hole punch was used to make a hole 20 mm away from the pathogen Phytophthora intolis. 200 μL of the above sterile supernatant was added, with 200 μL of sterile water as a blank control. After incubation at 28 °C for 5 days, the growth diameter of Phytophthora intolis was measured and the inhibition rate was calculated.

[0064] A two-part plate was used to prepare PDA-ISP2 solid medium. Strain GXMU-J15 was streaked onto one side of the ISP2 solid medium and incubated at 28°C for 3 days. Then, *Phytophthora nicotineae* spores were inoculated along the edge of one side of the PDA solid medium using a 7.5 mm punch. Using a two-part plate inoculated only with *Phytophthora nicotineae* as a control, the growth diameter of *Phytophthora nicotineae* was measured after incubation at 28°C for 7 days, and the inhibition rate was calculated.

[0065] The sterile supernatant with the best antibacterial effect was added to PDA medium at a ratio of 10% (v / v). The *Phytophthora indicum* cake was inoculated in the center of the plate. Plates with an equal amount of blank liquid medium were used as controls. After incubation at 28°C for 5 days, the growth diameter of *Phytophthora indicum* was measured, and the antibacterial rate was calculated.

[0066] The sterile supernatant with the best antibacterial effect was extracted three times with ethyl acetate at a 1:1 (v / v) ratio. The combined ethyl acetate extracts were concentrated to dryness under reduced pressure at 40°C to obtain the crude extract of strain GXMU-J15. The crude extract was dissolved in methanol at a concentration of 5 mg / mL, and the plate confrontation method was used. With methanol as a blank control, the growth diameter of Phytophthora tobaccois was measured after incubation at 28°C for 7 days, and the inhibition rate was calculated.

[0067] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%; The effects of strain GXMU-J15 treatment on the mycelial growth of Phytophthora tobaccois are shown in Table 3 and Figure 8As shown, different treatment solutions exhibited varying degrees of inhibitory effects. Strain GXMU-J15 exhibited the strongest antibacterial activity in ISP2 medium, with an inhibition rate of 46.92%. However, the toxic plate containing 10% (v / v) ISP2 fermentation broth significantly inhibited the growth of *Phytophthora indicum*, with a colony diameter of only 29.49 mm, significantly lower than the control group, resulting in an inhibition rate of 60.27%. The volatile gas antibacterial activity of strain GXMU-J15 was weak, at only 7.81%. A crude extract at a concentration of 5 mg / mL showed a significant inhibitory effect on *Phytophthora indicum*, with an inhibition rate of 77.19%. In summary, the experimental results indicate that strain GXMU-J15 has a significant antagonistic effect on *Phytophthora indicum*, causing problems such as slow mycelial growth and difficulty in expansion.

[0068] Table 3. Effects of sterile supernatant from strain GXMU-J15 cultured on different culture media on the mycelial growth of Phytophthora tobaccois (n=3)

[0069] Example 5

[0070] Preparation of fermentation broth of strain GXMU-J15

[0071] The strain GXMU-J15 was inoculated into ISP2 culture medium and cultured under the following conditions: 200 rpm, 28℃, and shaker fermentation for 7 days. The concentration of the obtained fermentation broth was 1×10⁻⁶. 8 CFU / mL.

[0072] Example 6

[0073] Growth-promoting effect of strain GXMU-J15

[0074] (1) Strain GXMU-J15 was inoculated onto functional media containing β-glucanase, cellulase, amylase, caseinase, and chitinase, and cultured at 28°C for 7 days. For β-glucanase and cellulase, 0.1% (w / v) Congo red was used for staining; for starch medium, iodine solution was used for staining. The appearance of a transparent area around the strain indicates the presence of enzyme activity. Spore suspension of strain GXMU-J15 was inoculated into ADF liquid medium and cultured at 28°C with shaking at 200 rpm for 7 days. The turbidity of the medium indicated the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity.

[0075] (2) Streak strain GXMU-J15 on Ashby medium and observe whether colonies are produced to determine its nitrogen fixation ability; inoculate strain GXMU-J15 on siderophore (CAS), glycine, Monkina inorganic phosphorus medium (PKO) and Monkina organic phosphorus medium, observe color changes and whether there are transparent areas to determine its siderophore, HCN and phosphorus solubilization abilities.

[0076] (3) The strain GXMU-J15 was inoculated into ISP2 liquid medium containing 100 mg / L L-tryptophan and cultured at 28°C and 200 rpm for 7 days. The supernatant was mixed with Salkowski colorimetric solution in equal proportion. IAA standard containing 50 μg / mL and blank medium were used as positive and negative controls. The pink appearance indicated the IAA yield.

[0077] The potential growth-promoting effects of strain GXMU-J15 on plants, such as Figure 9 As shown, strain GXMU-J15 produced clear regions of proteases, amylases, chitinases, β-glucans, and cellulases on various functional plates and could grow in ACC, indicating that strain GXMU-J15 has the ability to produce the corresponding enzymes. Simultaneously, strain GXMU-J15 also produced an orange-yellow halo on Chromium Blue-Blue Phenol S (side carrier) assay medium, a clear zone on calcium phytate medium, turned red upon addition of Salkowski colorimetric solution, showed no color change on glycine medium, could not grow on Ashby medium, and did not produce a clear zone on Pikovskaya medium, indicating that strain GXMU-J15 has the ability to produce side carriers and IAA, and to dissolve organic phosphorus, but not the ability to dissolve inorganic phosphorus or fix nitrogen.

[0078] Example 7

[0079] Growth-promoting effect of strain GXMU-J15 in potted plants

[0080] Tobacco seeds were planted in sterilized nutrient substrate soil and germinated in the dark. Healthy seedlings with similar growth at the 5-leaf stage were selected and transplanted into plastic round pots (12.5cm in diameter) filled with sterilized soil (nutrient substrate soil: loess = 1:1) for 5 days to allow them to recover. Each pot was irrigated with 1×10 [units of soil]. 8 40 mL of CFU / mL strain GXMU-J15 fermentation broth was applied every 7 days for a total of 4 treatments. Normal watering was maintained during this period. Tobacco seedlings treated with root irrigation water served as a control. Each treatment consisted of 3 tobacco seedlings, and each treatment was replicated 3 times. After 28 days, the plant height, stem diameter, root length, and fresh and dry weight of the above-ground and underground parts were measured.

[0081] The growth of tobacco in the experimental and control groups is shown in Table 4 and... Figure 10As shown, compared with the control, the application of strain GXMU-J15 significantly altered tobacco growth parameters and promoted tobacco growth. Strain GXMU-J15 significantly increased tobacco plant height, stem diameter, and the fresh and dry weight of the aboveground parts of the tobacco plants, with increases of 34.28%, 25.27%, 80.90%, and 50.07%, respectively. Similarly, strain GXMU-J15 significantly promoted root development. While there was no significant difference in root length, the fresh and dry weight of the roots were significantly higher than the control, with increases of 91.18% and 37.5%, respectively. Therefore, strain GXMU-J15 can promote root development, enabling plants to better absorb nutrients. The root-to-shoot ratio and seedling vigor index of tobacco plants treated with strain GXMU-J15 were 0.085 and 4.18, respectively, higher than the 0.081 and 2.18 of the control.

[0082] Table 4. Tobacco growth in the experimental and control groups (n=9)

[0083] Example 8

[0084] Disease control effect of strain GXMU-J15 in potted plants

[0085] Seedlings were raised using the same method as in Example 7. Fermentation broth of strain GXMU-J15, 58% metalaxyl-mancozeb, Bacillus subtilis agent, and Phytophthora tobaccois fermentation broth were applied by root injury and root irrigation. Four treatment groups were set up: (1) Treatment 1: 30 mL / plant of clean water was applied; (2) Treatment 2: 30 mL / plant of 500-fold diluted 58% metalaxyl-mancozeb solution (w / v) was applied; (3) Treatment 3: 1000-fold diluted commercially available Bacillus subtilis agent (100 billion / g, adjusted to 1×10⁻⁶ with sterile water) was applied. 8 (CFU / mL) 30mL / plant; (4) Treatment 4: 1×10 8 30 mL / strain of fermentation broth of strain GXMU-J15 with CFU / mL was applied to all treatments. 48 h after application, 1×10⁻⁶ strains were then applied. 7 30 mL / plant of *Phytophthora indicum* fermentation broth (cFU / mL, v / v). Eight seedlings were used in each treatment group, with each treatment replicated three times. The number of diseased plants and severity were assessed every 7 days post-inoculation. *Phytophthora indicum* disease severity grading was based on GB / T23222-2008. Disease incidence = (total number of plants - number of diseased plants) / total number of plants × 100%; Disease index = ∑(disease grade × number of plants at that disease grade) / (maximum disease grade × total number of plants) × 100%; Control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%. Results are shown in Table 5 and... Figure 11 As shown.

[0086] The GXMU-J15 strain showed a significant difference in the prevention of tobacco black shank. Seven days after inoculation with *Phytophthora indica*, no obvious yellowing of leaves was observed in tobacco plants; however, some plants developed lesions on their stems and wilted. Within 14-21 days, however, obvious yellowing and wilting of leaves appeared, leading to the death of the entire plant. Throughout the culture period, compared with the control group, tobacco plants in the 58% metalaxyl-mancozeb group and the *Bacillus subtilis* inoculant group grew poorly, while tobacco plants in the GXMU-J15 group grew well. After 21 days of culture, the disease incidence rate of tobacco plants in the GXMU-J15 group was 37.50%, significantly lower than the 95.83% in the control group. Furthermore, the disease index significantly decreased to 28.24, showing a significant decrease compared to the control group and the *Bacillus subtilis* inoculant group, but not as significant as the 58% metalaxyl-mancozeb group. The potted plant control efficacy of the *Bacillus subtilis* inoculant group against tobacco black shank was not significantly different from that of the GXMU-J15 group within 7 days. P The percentages of the strains exceeding 0.05% were all above 80%, gradually decreasing to 65.92%. This strain showed significantly higher efficacy against tobacco black shank than the control group, demonstrating remarkable application potential in the prevention and control of tobacco black shank.

[0087] Table 5. Growth of tobacco under different treatments (n=24)

[0088] As can be seen from the above embodiments, the present invention isolates and screens a strain of *Streptomyces brownii* (…). Streptomyces sp. GXMU-J15, a type of Streptomyces brownii, exhibits significant broad-spectrum antibacterial activity against various plant pathogens, demonstrating the ability to slow pathogen infection and reduce plant disease incidence. Streptomyces brownii GXMU-J15 showed an inhibition rate of 78.99% against Phytophthora tobaccoii, with an antibacterial activity of 60.27% in a 10% fermentation broth. It also increased tobacco plant height, stem diameter, and the fresh and dry weights of the aboveground and underground parts by 34.28%, 25.27%, 80.90%, 50.07%, 91.18%, and 37.5%, respectively. This was achieved at a pathogen concentration of 1×10⁻⁶. 7 The control effect of indoor potted plants at CFU / mL is over 65.92%; it can also cause the ends of Phytophthora tobacco hyphae to swell and branch, destroy the cell wall structure, thereby inhibiting the growth of Phytophthora tobacco hyphae, and can be applied to the control of tobacco black shank disease.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of brown Streptomyces ( Streptomyces sp.) GXMU-J15, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 20, 2023, with accession number GDMCC No:63901.

2. The *Streptomyces brownii* as described in claim 1 ( Streptomyces Application of sp.) GXMU-J15 in the preparation of products for preventing and controlling plant diseases caused by fungi.

3. The application according to claim 2, characterized in that, The fungi include Phytophthora nicotineae ( Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunata ), Fusarium graminearum ( Fusarium pseudograminearum ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. cubense One or more of the following.

4. The application according to claim 2, characterized in that, The product includes any one of fermentation broth, sterile supernatant, and crude fermentation extract.

5. A brown Streptomyces ( Streptomyces The fermentation broth of sp. GXMU-J15 is characterized by, The fermentation broth is composed of *Streptomyces brownii* as described in claim 1. Streptomyces It was prepared by sp.) GXMU-J15.

6. A brown Streptomyces ( Streptomyces The method for preparing fermentation broth of sp. GXMU-J15 is characterized by, Includes the following steps: The brown Streptomyces as described in claim 1 ( Streptomyces GXMU-J15 sp. was inoculated into the culture medium and cultured at 25-37℃ for 1-7 days to obtain the fermentation broth. The rotation speed during cultivation is 150~250 rpm; The culture medium is ISP2 liquid medium.

7. The application of the fermentation liquid according to claim 5 or the fermentation liquid prepared by the preparation method according to claim 6 in preventing and controlling plant diseases caused by fungi and improving plant growth performance; The fungi include Phytophthora nicotineae ( Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunata ), Fusarium graminearum ( Fusarium pseudograminearum ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. cubense One or more of the following; The plant in question is tobacco.

8. A brown Streptomyces ( Streptomyces The sterile supernatant of sp.) GXMU-J15, characterized in that, The method for preparing the sterile supernatant is as follows: Centrifuge the fermentation broth according to claim 5 for 8-12 minutes, take the supernatant, filter the supernatant, take the filtrate, and obtain a sterile supernatant. The centrifuge speed is 10000~14000 rpm; The filtration is membrane filtration, and the pore size of the membrane is 0.2~0.24μm.

9. The application of the sterile supernatant according to claim 8 in preventing and controlling plant diseases caused by fungi and improving plant growth performance; The fungi include Phytophthora nicotineae ( Phytophthora nicotianae ), basal root bead mold ( Berkeleyomyces basicola Staphylococcus aureus ( Botryosphaeria dothidea ), Banana anthrax bacteria ( Colletotrichum musae ), Curvularia crescentis ( Curvularia lunata ), Fusarium graminearum ( Fusarium pseudograminearum ) and Fusarium oxysporum Cuban-specific type ( Fusarium oxysporum f.sp. cubense One or more of the following; The plant in question is tobacco.

10. A brown Streptomyces fungus for controlling plant diseases caused by fungi ( Streptomyces The crude fermentation extract of sp. GXMU-J15, characterized in that, The method for preparing the fermented crude extract is as follows: The sterile supernatant of claim 8 was extracted with ethyl acetate 2 to 4 times. The extracts were combined and concentrated to dryness under reduced pressure to obtain the crude fermentation extract. The volume ratio of acetic acid and ethyl ester in the ethyl acetate is 1~2:1~2; The temperature for vacuum concentration is 38~42℃.

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