Staphylococcus Z8 for antagonizing pathogenic bacteria of mulberry bacterial wilt and application of staphylococcus Z8

Abstract

The invention discloses staphylococcus Z8 for antagonizing pathogenic bacteria of mulberry bacterial wilt and application of the staphylococcus Z8. The Z8 is identified as staphylococcus sp. And is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No.66232, and the preservation time is April 28, 2025. The invention further discloses a preparation method of the staphylococcus Z8 for antagonizing the pathogenic bacteria of mulberry bacterial wilt and application of the staphylococcus Z8 for antagonizing the pathogenic bacteria of mulberry bacterial wilt. The strain Z8 disclosed by the invention can destroy the formation of a biological membrane of the mulberry ralstonia solanacearum, so that the pathogenicity of the ralstonia solanacearum is reduced, and the strain Z8 can be used for preventing and treating the mulberry bacterial wilt. In addition, the strain Z8 does not affect the growth and development of the mulberry, the safety of the strain as a biocontrol strain is embodied, and the root activity and the total chlorophyll content can be improved. An efficient and environment-friendly microbial resource is provided for biological prevention and control of mulberry bacterial wilt, traditional chemical pesticides can be replaced, and environmental pollution is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microbial control, and in particular to Staphylococcus aureus Z8 antagonistic to mulberry bacterial wilt pathogens and applications thereof. Background Art

[0002] Mulberry trees are a traditional tree species with both economic and ecological value, and are an important foundation for the development of the sericulture industry. In recent years, with the rapid development of the sericulture industry and the promotion of intensive management models, mulberry tree diseases have become increasingly prominent, causing significant economic losses and becoming a major factor hindering the green and sustainable development of the sericulture industry. Among them, mulberry bacterial wilt is a soil-borne bacterial disease caused by Ralstonia pseudosolanacearum. This disease occurs in all major mulberry-growing areas in my country, causing devastating damage to the mulberry industry. Ralstonia pseudosolanacearum typically invades the host plant roots, enters the xylem vessels, and begins to proliferate rapidly. Its high bacterial density and the exopolysaccharides (EPS) it secretes clog the vascular system, impairing water transport and causing irreversible plant wilt. Early symptoms include brown streaks in the xylem, loss of luster, curling, discoloration, and wilting of leaves. As the disease progresses and spreads, the entire plant's xylem turns brown and black, pus oozes from the cuts of diseased roots or branches, and large areas of leaves wilt and wither. In severe cases, the roots of mulberry seedlings dry up and rot, and all leaves fall off. This disease, also known as "mulberry cancer," is a major impediment to the sustainable development of the sericulture industry.

[0003] Currently, the prevention and control of mulberry bacterial wilt mainly relies on chemical control measures, but these traditional control methods have drawbacks such as drug resistance. The long-term use of chemical fungicides not only kills beneficial microorganisms in the soil and reduces microbial diversity, but can also cause drug residues, damage the ecological environment, and affect silkworm breeding. In comparison, biological control technologies based on microbial antagonism are not only environmentally friendly but also effectively protect crop health and reduce environmental pollution. Therefore, the development of environmentally friendly and efficient biological control methods has become a focus of current green disease control research. However, current research on biocontrol bacteria for mulberry bacterial wilt is relatively insufficient. Existing work has mostly focused on the screening and identification of endophytic bacteria in plants. In addition, the relative scarcity of biocontrol bacteria resources has hindered the improvement of field control effectiveness. Therefore, the discovery of new biocontrol bacteria is of great significance for improving the prevention and control of mulberry bacterial wilt.

[0004] Research on the use of Staphylococcus as a biocontrol agent in plant disease control is relatively limited, but some studies have explored the potential application of Staphylococci or their metabolites in plant disease control. As biocontrol agents against plant pathogens, Staphylococci can inhibit plant pathogens, such as the partially coagulase-negative Staphylococci (CoNS) S. epidermidis and S. saprophyticus, by producing antimicrobial peptides or other metabolites, and can also inhibit the growth of plant pathogens. Regarding plant health promotion, some Staphylococci may enhance plant defenses against pathogens by competing for ecological niches or inducing plant systemic resistance (ISR), similar to the mechanism of action of plant growth-promoting rhizobacteria (PGPR). However, there are currently no reports on the use of Staphylococcus sp. in controlling mulberry bacterial wilt. Therefore, investigating the antibacterial activity of Staphylococci against R. solanacearum and its mechanism of inhibition could provide new insights into the development of biocontrol agents for mulberry bacterial wilt in production. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a mulberry rhizospheric Staphylococcus Z8, which has a broad-spectrum antibacterial property. The Staphylococcus Z8 of the present invention has a good antagonistic effect on the pathogen of mulberry bacterial wilt (Ralstonia pseudosolanacearum), with an inhibition rate of more than 50%. It can be used for the prevention and treatment of mulberry bacterial wilt and has broad application prospects.

[0006] Another object of the present invention is to provide a biocontrol preparation of Staphylococcus aureus Z8 that antagonizes the pathogen of mulberry bacterial wilt and its application.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the Staphylococcus Z8 described in the present invention has been identified as Staphylococcus sp. and has been deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No. 66232. The deposit time is April 28, 2025, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, No. 59, Compound 100, Xianlie Middle Road, Guangzhou, Postal Code: 510070.

[0008] The Staphylococcus Z8 strain was isolated from the rhizosphere soil of healthy mulberry trees by a dilution coating method, and was identified as Staphylococcus by morphology and molecular biology and named Staphylococcus Z8.

[0009] The invention relates to the use of Staphylococcus aureus Z8 in inhibiting or antagonizing bacterial wilt pathogens.

[0010] Wherein, the bacterial wilt pathogen is Ralstonia pseudosolanacearum.

[0011] The Staphylococcus aureus Z8 is used to inhibit or antagonize bacterial wilt by inhibiting the growth of bacterial wilt, destroying the bacterial morphology of bacterial wilt, reducing the biofilm formation ability, and destroying the integrity and continuity of the bacterial wilt biofilm.

[0012] Among them, the Staphylococcus aureus Z8 reduces the motility of Ralstonia solanacearum and is used in inhibiting or antagonizing Ralstonia solanacearum.

[0013] Furthermore, a plate-based antibacterial test revealed a 50% inhibition rate against Ralstonia solanacearum. The active fermentation broth not only significantly inhibited the growth of Ralstonia solanacearum and reduced its biofilm formation ability, but also disrupted the integrity and continuity of the biofilm. Furthermore, it reduced the bacterial ring size, decreased motility, and destroyed the Ralstonia solanacearum cells.

[0014] The Staphylococcus aureus Z8 described in the present invention can inhibit or antagonize the growth of mulberry bacterial wilt pathogen, and at the same time improve the POD activity, root activity and total chlorophyll content of the treated mulberry seedlings, and is used in disease prevention and growth promotion.

[0015] Furthermore, the Staphylococcus aureus Z8 has little effect on the growth of mulberry seedlings themselves, and has a good function of antagonizing mulberry solanacearum, and can be used to prepare mulberry solanacearum biocontrol preparations, and has good application prospects.

[0016] The invention comprises a biocontrol preparation of the Staphylococcus Z8 strain or its fermentation liquid.

[0017] The preparation method of the biocontrol agent comprises activating the Staphylococcus aureus strain Z8 in NA medium, inverting the culture medium in a constant temperature incubator, and then, after the bacteria have fully grown, picking a small amount of bacteria and fermenting them in NB medium. The fermentation liquid is filtered to obtain the filtrate, which is the fermentation liquid of Staphylococcus aureus Z8.

[0018] Use of the Staphylococcus Z8 of the present invention or the biocontrol agent according to claim 8 in preventing and controlling mulberry bacterial wilt.

[0019] Use of the Staphylococcus Z8 of the present invention or the biocontrol agent according to claim 8 in promoting the growth of mulberry trees.

[0020] The method for isolating and identifying Staphylococcus aureus Z8 of the present invention comprises the following steps:

[0021] (1) The bacterial strain Z8 was isolated from the rhizosphere soil of healthy mulberry trees by the dilution coating method.

[0022] (2) Using molecular biological methods, phylogenetic analysis was constructed based on 16s rRNA gene sequences, such as Figure 2 shown.

[0023] (3) The morphological characteristics of Staphylococcus aureus Z8 described in the present invention are as follows: strain Z8 grows rapidly and can produce colonies after 24 hours of constant temperature culture at 28°C. The colonies are yellow, nearly round, with a diameter of 1-3 mm, neat edges, and a smooth and moist surface.

[0024] The present invention also includes the antibacterial mechanism of Staphylococcus aureus Z8 against Ralstonia solanacearum, which includes the plate antibacterial effect of Z8 on Ralstonia solanacearum, the influence of active fermentation liquid on the growth curve of Ralstonia solanacearum, and the influence on biofilm formation and motility of Ralstonia solanacearum.

[0025] The Staphylococcus aureus Z8 of the present invention does not have any inhibitory effect on seed germination and plant growth rate, but can improve root activity and total chlorophyll content, and can be used as a feasible biocontrol agent.

[0026] The present invention identifies a strain of bacteria, Z8, with a strong antagonistic effect against Ralstonia solanacearum. Morphological and molecular biological identifications indicate that the strain is a Staphylococcus aureus. Z8 also demonstrated an antibacterial activity of 50% against Ralstonia solanacearum. Z8 can disrupt the biofilm of Ralstonia solanacearum, reduce SOD enzyme activity, increase biofilm permeability, and inhibit the motility of Ralstonia solanacearum. This, to a certain extent, clarifies the antibacterial mechanism of Z8 against Ralstonia solanacearum. Furthermore, Z8 does not affect the growth and development of mulberry trees themselves, but instead increases POD enzyme activity, root vitality, and chlorophyll content, demonstrating its safety and effectiveness as a biocontrol strain.

[0027] The present invention separates and purifies a strain of bacteria from the root secretions of healthy mulberry trees by a dilution coating method. It is identified as Staphylococcus by morphology and molecular biology and is named Z8. Its biocontrol potential is evaluated by plate inhibition experiment, and it is verified that the strain has an inhibitory effect on mulberry bacterial wilt MRS-5. Further, by measuring the effect of the active fermentation liquid of Z8 bacterial strain on the swimming motility, biofilm formation and biofilm permeability of mulberry bacterial wilt MRS-5, a preliminary exploration of the biocontrol mechanism of strain Z8 is made, in order to clarify the biocontrol mechanism of Staphylococcus Z8, and provide a certain scientific basis for the development of eco-type microbial source agents for preventing and treating mulberry bacterial wilt. In summary, the research and development of Staphylococcus is of great significance for preventing and treating mulberry bacterial wilt. At present, there are limited reports on the relevant technologies of Staphylococcus, and there is no biocontrol agent derived from Staphylococcus for preventing and treating mulberry bacterial wilt on the market, which is still a technical gap.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] Through isolation and screening, the present invention identified a Staphylococcus Z8 strain that has a strong inhibitory effect on mulberry bacterial wilt and is highly safe for mulberry growth, and preliminarily elucidated its antibacterial mechanism, which can provide a theoretical basis for exploring the subsequent biocontrol application potential of Staphylococcus Z8 strain.

[0030] The Staphylococcus aureus Z8 of the present invention can be used to prepare a novel biological control agent for Ralstonia solanacearum. It does not inhibit seed germination or plant growth rate, effectively solving the soil pollution caused by the use of traditional chemical agents for Ralstonia solanacearum, and is highly effective and environmentally friendly. The biological control agent of the present invention can be produced using fermentation equipment commonly used in the fermentation industry, and has the advantages of low production cost, ease of use, and good antibacterial and control effects. The present invention is of great significance for protecting the ecological environment and reducing chemical pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : This is a graph showing the growth morphology of the Staphylococcus aureus Z8 strain on NA culture medium in the present invention, wherein A is the colony morphology and B is the Gram staining result.

[0032] Figure 2 This is a phylogenetic tree of the Z8 strain constructed based on the 16s rRNA gene amplified sequence in one of the embodiments of the present invention.

[0033] Figure 3 This is a diagram showing the antagonistic effect of Staphylococcus aureus Z8 on Ralstonia solanacearum MRS-5 in one of the examples of the present invention.

[0034] Figure 4 This is the effect of the active fermentation broth of Staphylococcus aureus Z8 on the growth curve of Ralstonia solanacearum MRS-5 in one of the examples of the present invention.

[0035] Figure 5 This is the effect of Staphylococcus aureus Z8 on the SOD enzyme activity and biofilm formation of Ralstonia solanacearum MRS-5 in one of the examples of the present invention.

[0036] Figure 6 This is a scanning electron micrograph of the inhibitory effect of Staphylococcus aureus Z8 on the growth of Ralstonia solanacearum MRS-5 in one embodiment of the present invention. Figure A is a scanning electron micrograph of normal Ralstonia solanacearum MRS-5, and Figure B is a scanning electron micrograph of Ralstonia solanacearum MRS-5 after being treated with the active fermentation liquid of Staphylococcus aureus Z8.

[0037] Figure 7 This is the effect of Staphylococcus aureus Z8 on the motility of Ralstonia solanacearum MRS-5 in one of the examples of the present invention.

[0038] Figure 8This is the effect of Staphylococcus aureus Z8 on the growth of mulberry seedlings in one embodiment of the present invention, wherein Figure A shows the effect of Staphylococcus aureus Z8 on the growth of mulberry seedlings, and Figures B, C, and D respectively show the changes in POD, mulberry root activity, and total chlorophyll content of mulberry seedlings after treatment with Staphylococcus aureus Z8. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and examples.

[0040] Strain: Ralstonia pseudosolanacearum MRS-5 is a wild-type Ralstonia solanacearum provided by Jiangsu University of Science and Technology. The Ralstonia pseudosolanacearum MRS-5 is deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC 1.3250 (a common non-patented deposit). The deposit date is June 6, 2022. The applicant has already mentioned it in the prior published patent.

[0041] Mulberry seeds: Fengchi mulberry seeds are preserved by the Mulberry Science Research Center of Jiangsu University of Science and Technology / Chinese Academy of Agricultural Sciences.

[0042] Sources of experimental reagents and equipment:

[0043] Experimental reagents: Glucose, yeast powder, beef extract, polypeptone, sucrose, agar powder, glucose, phenol, anhydrous ethanol and isoamyl alcohol were purchased from Sinopharm Group, and RNase A, Tris-HCl and other reagents were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0044] Experimental equipment: -80℃ refrigerator (WiseCryo, Korea), high-speed refrigerated centrifuge (Thermo FRESCO17, Thermo Fisher, America), SpectraMaxi3 microplate reader (BUCHI, Switzerland), ultra-clean workbench (Zhejiang Fuxia Medical Technology Co., Ltd.), digital display gas bath constant temperature oscillator THZ-82A (Nanjing Lisi Gao Instrument Equipment Co., Ltd.), high pressure sterilizer (Zhiwei Instrument), constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), light incubator (Nanjing Sanshi Biotechnology Co., Ltd.), biological microscope (Nikon ECLIPSE Si RS).

[0045] Solution and test culture medium configuration:

[0046] The composition of NA medium is as follows: beef extract 3g, yeast powder 1g, polypeptone 5g, sucrose 1g, agar powder 15g, distilled water to 1000mL, 121℃, and sterilized for 20min.

[0047] The composition of NB medium is as follows: 3 g beef extract, 1 g yeast powder, 5 g polypeptone, 1 g sucrose, 1000 mL distilled water, sterilized at 121°C for 30 min.

[0048] The composition of NA semi-solid culture medium is as follows: based on the above NA culture medium, the agar powder content is reduced by half.

[0049] The composition of NB medium is as follows: the above-mentioned NA medium is not added with agar powder.

[0050] Example 1

[0051] Soil samples were collected from the base of China Sericulture Research Institute in Zhenjiang, Jiangsu Province. 2 g of sample was added to 98 mL of sterile water to prepare a soil suspension. After vibrating and mixing thoroughly for 24 h, a 10-fold dilution method was used to dilute the suspension into 10 -3 , 10 -4 , 10 -5 Then, take 100 μL of the soil dilution and evenly spread it on the NA medium, invert it and culture it in a constant temperature incubator at 28°C, and observe it regularly. After the suspected bacteria grow on the NA plate, immediately use a sterile inoculation loop to pick a single colony and transfer it to a new NA plate for purification. Then, inoculate the purified strain into NB medium and culture it in a constant temperature shaker at 28°C and 180 rpm for 24 hours. Store the test tube culture in a 4°C refrigerator for short-term storage. At the same time, take 700 μL of bacterial solution and add it to the cryopreservation tube, then add 700 μL of 50% glycerol and mix well. After quick freezing in liquid nitrogen, store it in a -80°C refrigerator for long-term storage.

[0052] Example 2

[0053] Preparation of bacterial suspensions of strain Z8 and Ralstonia solanacearum MRS-5

[0054] A strain designated Z8, stored at -80°C, was activated with 100 μL of glycerol stock in NA medium. The NA plate was inverted and incubated at 28°C for 24 hours. Using a sterile operating table, a Z8 colony was picked with an inoculating loop and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized NB liquid medium. The culture was incubated at 28°C and shaken at 180 rpm for 12 hours to obtain the seed solution. A 1.00% inoculum volume was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterilized NB medium. The culture was then incubated at 28°C and 180 rpm for 48 hours in a shaker. The fermentation broth was then transferred to a sterile 50 mL centrifuge tube and centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant was sterilized by filtration through a 0.22 μm filter and stored at 4°C until further use. This was the active Z8 fermentation broth.

[0055] MRS-5 was cultured in CPG medium using the three-line method. After culturing in a constant temperature incubator at 28°C for 24 hours, a single colony was picked and inoculated into CPG liquid medium. A single colony was picked and inoculated into CPG liquid medium at 28°C and 180 rpm until the OD 600 =0.2, which is the MRS-5 bacterial suspension of Ralstonia solanacearum.

[0056] Example 3

[0057] Determination of the antagonistic effect of Z8 on Ralstonia solanacearum MRS-5

[0058] The antibacterial test was carried out using the plate confrontation method ( Figure 3 ), 5 mL of the MRS-5 bacterial suspension of Ralstonia solanacearum prepared in Example 3 was mixed with 100 mL of pre-cooled, unsolidified NA culture medium, and then poured into the middle of the plate with a sterile hole puncher to make a small hole for later use. 20 μL of the active fermentation liquid of bacteria Z8 was added dropwise to the center of the small hole of the NA culture medium plate containing Ralstonia solanacearum. 100 mL of pre-cooled, unsolidified NA culture medium with sterile water added was used as a control. Three replicates were set for each treatment, and observations were made every day, and the size of the inhibition zone was measured using the cross-cross method. According to the diameter of the inhibition zone of the bacteria on the third day of inoculation, it was calculated that the inhibition rate of Staphylococcus aureus Z8 against Ralstonia solanacearum was 50%, and the results are shown in Table 1.

[0059] Table 1 Antibacterial effect of Staphylococcus aureus Z8 on Ralstonia solanacearum MRS-5

[0060]

[0061] Example 4

[0062] Bacterial morphological identification and molecular biological identification

[0063] (1) Morphological identification

[0064] The morphological characteristics of the strain Z8 screened by the present invention after being cultured on NA plates for 24 hours are shown in FIG. Figure 1 As shown in A. The strain grows rapidly and can form colonies after 24 hours of constant temperature cultivation at 28°C. The colonies are yellow, nearly round, 1-3 mm in diameter, with neat edges and a smooth and moist surface.

[0065] (2) Molecular biological identification

[0066] PCR amplification was performed using 16S rRNA gene primers synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0067] The 16S rRNA gene primer sequences are as follows:

[0068] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0069] 1429R:5'-GGTTACCTTGTTACGACTT-3'

[0070] The PCR reaction system was 25 μL: 12.5 μL 2×T5Super PCR Mix (Qingke Biotechnology), 1.5 μL template DNA, 1 μL each of forward and reverse primers (10 μmol / L), and ddH2O was added to make up to 25 μL.

[0071] PCR amplification reaction program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, 35 cycles; final extension at 72°C for 2 min.

[0072] The PCR product was verified by 1% agarose gel electrophoresis and then sent to Shanghai Sangon Biotechnology Co., Ltd. for Sanger sequencing. The sequencing result is shown in SEQ ID NO.1. The sequence obtained was compared online on the website of the National Center for Biotechnology Information (NCBI). At the same time, based on the obtained sequence and other highly homologous strain-related sequence information obtained from GenBank, a phylogenetic tree was constructed using the Neighbor-Joining calculation method of MEGAX software to clarify the taxonomic status of the strain. The results showed that the 16s rRNA amplified sequence of the strain was stably clustered in the same branch with strains of the genus Staphylococcus, and its phylogenetic relationship was closest to PP948657.1Staphylococcus sp. strain LH-403 ( Figure 2 Based on its morphological, physiological, and biochemical characteristics, the strain was named Staphylococcus sp. Z8, or simply Staphylococcus Z8. The deposit number is GDMCC No. 66232; the depositor is the Guangdong Provincial Microbiological Culture Collection; the address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, 510070, China; the date of deposit is April 28, 2025.

[0073] Example 5

[0074] Effects of Staphylococcus Z8 active fermentation broth on the growth curve of Ralstonia solanacearum MRS-5

[0075] By measuring the effect of Staphylococcus Z8 fermentation broth on the growth curve of MRS-5, its biocontrol and antibacterial mechanism can be further studied. Take 1mL of Staphylococcus Z8 active fermentation broth (Example 3) and add it to 50mL of NB liquid culture medium and mix evenly. Take 100μL of the MRS-5 solanacearum bacterial solution from Example 3 and mix it inoculate it into the same NB culture medium. Cultivate it in a shaker at 28℃ and 180rpm. Each treatment was repeated 3 times, and samples were taken every 2h to measure the OD value of each group of solanacearum bacterial solution. 600 By comparing the content of Ralstonia solanacearum between different treatment groups, the growth curve of Ralstonia solanacearum was drawn. The results showed that after treatment with Staphylococcus aureus Z8, Ralstonia solanacearum MRS-5 grew slowly, indicating that Z8 had a significant inhibitory effect on MRS-5 ( Figure 4 ).

[0076] Example 6

[0077] Effects of Staphylococcus Z8 active fermentation broth on SOD enzyme activity and biofilm formation of Ralstonia solanacearum MRS-5

[0078] Superoxide dismutase (SOD) has a dual effect on bacteria. It participates in the bacteria's own antioxidant defense system and may also influence their pathogenicity and interactions with the host. By regulating ROS signaling, it promotes bacterial biofilm formation (such as in Pseudomonas aeruginosa), enhancing drug resistance and persistent infection. Biofilms provide protection for bacteria, counteracting host immune defense mechanisms and increasing bacterial drug resistance. This study measured the biofilm formation ability and SOD activity of MRS-5 solanacearum after treatment with Staphylococcus aureus Z8. 800 μL of Z8 active fermentation broth (Example 3) and 200 μL of a solanacearum suspension (Example 3) were added to a 24-well plate, and NB medium was added to 1.5 mL. The 24-well plate was incubated in a 28°C constant temperature incubator for 24 hours. After incubation, the supernatant was removed and the plate was washed three times with 1× PBS buffer. 200 μL of 1% crystal violet was added for staining. After standing for 15 minutes, the plate was washed twice with PBS and dried at room temperature for 30 minutes. To quantify the biofilm biomass, 200 μL of 95% ethanol was added to each well to dissolve crystal violet. After 30 minutes, the absorbance at 595 nm was measured to reflect the formation of the R. solanacearum biofilm. Each treatment was repeated three times. The absorbance measurement showed that the amount of biofilm formed by Staphylococcus aureus Z8 and R. solanacearum MRS-5 after treatment was significantly reduced compared to the control group ( Figure 5 B). The results showed that treatment with Staphylococcus aureus Z8 could significantly reduce the pathogenicity of MRS-5 by destroying the integrity of the biofilm structure of Ralstonia solanacearum MRS-5. The total superoxide dismutase (hydroxylamine method) assay showed that the superoxide dismutase (SOD) activity of the treated MRS-5 strain showed a significant downward trend compared with the control group (P<0.05). Figure 5 A).

[0079] Example 7

[0080] Scanning electron microscopy images of the inhibitory effect of Staphylococcus aureus Z8 on the growth of Ralstonia solanacearum MRS-5

[0081] The cell slides were placed in a 12-well plate, and 800 μL of CPG medium and Ralstonia solanacearum liquid (OD 600 =0.6), cultured at 30°C for 12 h, and 200 μL of Staphylococcus aureus Z8 bacterial solution (OD 600 =0.6) to treat mulberry solanacearum MRS-5 (sterile water control) and continue to culture for 8h. The cultured cell slides were pre-fixed with pentanediol to observe the morphological changes of solanacearum and record images. The growth morphological changes of mulberry solanacearum MRS-5 after treatment with Staphylococcus aureus Z8 were observed by scanning electron microscopy. The surface structure of the solanacearum cells in the control group was intact and the morphology was full ( Figure 6 A). Compared with the control group, the morphology of the treated MRS-5 strains was severely deformed, the bacterial surface was blurred, and the dissolved substances accumulated as indicated by the red arrows in the figure ( Figure 6 B).

[0082] Example 8

[0083] Effects of Staphylococcus aureus Z8 active fermentation broth on the motility of Ralstonia solanacearum MRS-5

[0084] Motility is a flagella-mediated form of movement of Ralstonia solanacearum, which enables the bacteria to effectively invade the host, cause disease and colonize. The present invention explores the effect of bacterial strain Z8 treatment on the motility of Ralstonia solanacearum MRS-5. 2mL of Z8 active fermentation broth (Example 3) and 8mL of Ralstonia solanacearum MRS-5 bacterial suspension (Example 3) were placed in a shaker at 28°C and 180rpm for 48h, and the control group was treated with an equal amount of NB culture medium to treat Ralstonia solanacearum. 5μL of bacterial solution from different treatment groups was respectively drawn and dropped into NB semi-solid culture medium, and inverted in a constant temperature incubator at 28°C for 12h to observe the growth of Ralstonia solanacearum, and the diameter of the bacterial ring growth was measured by the cross-cross method, and each treatment was repeated 3 times. After treatment with Staphylococcus aureus Z8, the diameter of the bacterial ring formed on the semi-solid culture medium was significantly smaller than that of the control group ( Figure 7 A). The diameter of the bacterial ring was measured by the cross-cross method. The diameter of the colony after 12 hours of treatment minus the diameter of the colony in the control group was the colony growth diameter. Compared with the control group, the colony growth diameter of MRS-5 on mulberry wilt after Staphylococcus Z8 treatment was significantly reduced ( Figure 7 B).

[0085] Example 9

[0086] Growth-promoting effect of Staphylococcus aureus Z8 on mulberry trees

[0087] (1) Effects of Staphylococcus aureus Z8 on root growth of mulberry seedlings

[0088] The results of the above-mentioned embodiments show that Staphylococcus Z8 has an inhibitory effect on mulberry bacterial wilt MRS-5. In order to verify whether Staphylococcus Z8 will affect the growth of mulberry trees themselves, the present invention measures the total chlorophyll content, root activity and POD enzyme activity of mulberry seedlings after treatment with Staphylococcus Z8. The Fengchi mulberry seeds were disinfected with 75% alcohol for 1 minute and 5% NaClO solution for 5 minutes, and then soaked in Z8 active fermentation liquid (Example 3) for 5 hours. After absorbing the excess liquid on the surface of the seeds with sterile filter paper, they were placed in a culture dish with sterile filter paper on the bottom. The Z8 active fermentation liquid was used as the treatment group and the clean water group as the control, and the germination and growth conditions were recorded. The germination rate of each group of seeds and the root length of the mulberry seedlings were measured. Each group treated 20 seeds and set up 3 biological replicates. The results showed that the root length of the mulberry seedlings in the Staphylococcus Z8 treatment group was measured. Compared with the control group, the Z8 active fermentation liquid had a promoting effect on the germination of mulberry seeds and the growth of mulberry trees ( Figure 8 A).

[0089] (2) Effect of Staphylococcus Z8 fermentation broth on POD enzyme activity in mulberry seedlings

[0090] The growth-promoting effect of Staphylococcus Z8 can be further studied by measuring the effect of Staphylococcus Z8 fermentation liquid on the POD enzyme activity of mulberry seedlings. The guaiacol colorimetric method was used to determine the POD enzyme activity of seeds. 2 mL of Staphylococcus Z8 active fermentation liquid (Example 3) was mixed evenly with 5 mL of NB liquid medium. The mixed liquid was infiltrated into the roots of mulberry seedlings, and 7 mL of NB liquid medium was infiltrated into the control group seedlings. Each treatment was repeated 3 times, and the OD was measured. 470 By comparing the absorbance values ​​between different treatment groups, the tissue sample POD (U) was calculated using the following formula: 测定 1-A 测定 0)×V T} / (W×V S ×0.01×t). The peroxidase (POD) enzyme activity assay kit (Cat. No. R30312-50T) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The results showed that compared with the control group, the mulberry seedlings treated with Staphylococcus Z8 active fermentation liquid could significantly increase the POD enzyme activity of the mulberry seedlings ( Figure 8 B), further indicating that the active fermentation liquid of Staphylococcus aureus Z8 has a growth-promoting effect on mulberry seedlings.

[0091] (3) Effects of Staphylococcus Z8 fermentation liquid on root activity of mulberry seedlings

[0092] The effect of Staphylococcus Z8 fermentation liquid on the root activity of mulberry seedlings can be measured to further study the growth-promoting effect of Staphylococcus Z8. The root activity of seedlings was determined by TTC colorimetry. 2 mL of Staphylococcus Z8 active fermentation liquid (Example 3) was mixed evenly with 5 mL of NB liquid medium. The mixed liquid was used to infiltrate the roots of mulberry seedlings, and 7 mL of NB liquid medium was used to infiltrate the seedlings of the control group. Each treatment was repeated 3 times. 0.2-0.5 g of mulberry seedling fibrous roots were taken, washed, dried with filter paper, and completely immersed in TTC Assay Buffer working solution. Incubated at 37 ° C in the dark for 1-3 h, 2 mL of TTC stop solution was added to terminate the reaction. The roots of the control group were first completely immersed in 2 mL of TTC stop solution to kill the root sample, and then 10 mL of TTC Assay Buffer working solution was added and incubated at 37 ° C in the dark for 1-3 h. Remove the roots, dry them with filter paper, place them in a mortar or homogenizer, add 3-4 ml of ethyl acetate, grind or homogenize thoroughly to extract TTF, transfer the red extract (TTF) to a centrifuge tube, and wash the residue 2-3 times with a small amount of ethyl acetate, then transfer the washing liquid to a centrifuge tube, and finally add ethyl acetate to 10 mL and shake well. Measure the OD of each treatment group 485 The TTF content of the series of standard solutions (25, 50, 100, 150, 200 μg) was used as the horizontal axis, and the corresponding absorbance was used as the vertical axis to draw the TTF standard curve. The TTF content or TTC reduction amount (μg) of the test sample was calculated according to the regression equation, which was the root activity or dehydrogenase activity. The results showed that compared with the control group, the mulberry seedlings treated with the active fermentation liquid of Staphylococcus aureus Z8 could significantly improve the root activity of the mulberry seedlings ( Figure 8 C), further indicating that the active fermentation liquid of Staphylococcus aureus Z8 has a growth-promoting effect on mulberry seedlings.

[0093] (4) Effect of Staphylococcus Z8 fermentation broth on total chlorophyll content in mulberry seedlings

[0094] The growth-promoting effect of Staphylococcus Z8 can be further studied by measuring the effect of Staphylococcus Z8 fermentation liquid on the total chlorophyll content of mulberry seedlings. The chlorophyll content of seedlings was determined by colorimetry. 2 mL of Staphylococcus Z8 active fermentation liquid (Example 3) was mixed evenly with 5 mL of NB liquid culture medium. The mixed liquid was infiltrated into the roots of mulberry seedlings, and 7 mL of NB liquid culture medium was infiltrated into the control group seedlings. Each treatment was repeated 3 times, cultured for 7 days, and the OD value of chlorophyll in each group of mulberry seedlings was measured. 665 With OD 649 , calculate the chlorophyll A, chlorophyll B and total chlorophyll content ( Figure 8D). The kit for measuring chlorophyll content in mulberry (Cat. No. R30354-50T) was provided by Shanghai Yuanye Biotechnology Co., Ltd. The results showed that compared with the control group, the treatment of mulberry seedling roots with the active fermentation liquid of Staphylococcus aureus Z8 increased the chlorophyll A and total chlorophyll content of the seedlings ( Figure 8 D), further demonstrating that the active fermentation liquid of Staphylococcus aureus Z8 has a growth-promoting effect on mulberry seedlings. Therefore, Staphylococcus aureus Z8 can promote the growth of mulberry seedlings while having no inhibitory effect on the growth of mulberry trees themselves, making it a potential biocontrol resource for the development of MRS-5, a fungus that causes solanacearum wilt in mulberry trees.

[0095] In summary, through isolation and screening, the present invention identified a Staphylococcus aureus strain Z8 that has a strong inhibitory effect on the pathogen of mulberry bacterial wilt and is highly safe for mulberry trees, and to a certain extent clarified its antibacterial mechanism, providing a solid theoretical basis for in-depth exploration of the potential application value of Staphylococcus aureus Z8 strain in the field of biological control and green prevention and control of mulberry bacterial wilt.

Claims

1. A Staphylococcus aureus Z8 was identified as Staphylococcus sp. and has been deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No. 66232 and the deposit date is April 28, 2025.

2. Use of Staphylococcus aureus Z8 according to claim 1 in inhibiting or antagonizing bacterial wilt pathogens.

3. The use according to claim 2, characterized in that The bacterial wilt pathogen is preferably Ralstonia pseudosolanacearum.

4. The use according to claim 2, characterized in that The Staphylococcus aureus Z8 is used to inhibit or antagonize bacterial wilt by inhibiting the growth of bacterial wilt, destroying the bacterial morphology of bacterial wilt, reducing the biofilm formation ability, and destroying the integrity and continuity of the bacterial wilt biofilm.

5. The use according to claim 2, characterized in that The Staphylococcus aureus Z8 reduces the motility of Ralstonia solanacearum and is used in inhibiting or antagonizing Ralstonia solanacearum.

6. Use of the Staphylococcus aureus Z8 according to claim 1 in inhibiting or antagonizing the growth of mulberry bacterial wilt pathogen, while increasing the POD activity, root activity and total chlorophyll content of the treated mulberry seedlings in disease prevention and growth promotion.

7. A biocontrol agent comprising the Staphylococcus Z8 strain or a fermentation broth thereof according to claim 1.

8. The biocontrol agent according to claim 7, characterized in that The preparation method of the biocontrol agent comprises the following steps: activating the Staphylococcus aureus strain Z8 in NA culture medium, inverting the culture medium and culturing the culture medium in a constant temperature incubator; and after the bacteria have fully grown, picking a small amount of bacteria and fermenting them in NB culture medium; filtering the fermentation liquid to obtain the filtrate, which is the fermentation liquid of Staphylococcus aureus Z8.

9. Use of the Staphylococcus aureus Z8 according to claim 1 or the biocontrol agent according to claim 8 in preventing and controlling mulberry bacterial wilt.

10. Use of the Staphylococcus aureus Z8 according to claim 1 or the biocontrol agent according to claim 8 in promoting the growth of mulberry trees.

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