Bacillus subtilis Gxunw-30 and application thereof

Abstract

The invention discloses bacillus subtilis Gxunw-30 and application thereof, and belongs to the technical field of agricultural microorganisms. The bacillus subtilis Gxunw-30 provided by the invention is preserved in Guangdong Microbial Culture Collection Center on October 12, 2024, and the preservation number of the bacillus subtilis Gxunw-30 is GDMCC No: 65245. The bacillus subtilis Gxunw-30 provided by the invention has the capability of inhibiting plant pathogens in a broad spectrum, and can be used for antagonizing a variety of plant pathogens such as helminthosporium triticum, trichosporon pseudoacaceae, hechiense fusarium, fusarium chlamydosporum, cryphonectria parasitica, fusarium pseudograminearum, botryosphaeria dothidea and the like. The strain has the properties of decomposing organophosphorus, producing protease, producing ammonia, producing indole and fixing nitrogen, and has the biological properties of plant growth promotion and the like. The recombinant bacterium also contains a gene for coding an antibacterial substance, and has the capability of synthesizing the antibacterial peptides of iturin, surfactin, fengmycin and bacilysin, and the recombinant bacterium also contains a gene for coding the antibacterial substance and has the capability of synthesizing the antibacterial peptides of iturin, surfactin, fengmycin and bacilysin. The strain has the effects of preventing diseases and promoting growth, provides a good biocontrol resource for prevention and treatment of plant diseases, and has a relatively good application value for research and development of plant growth-promoting preparations and biocontrol microbial agents.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to Bacillus subtilis Gxunw-30 and applications thereof. Background Art

[0002] As a basic industry of the national economy, agriculture plays a vital role in economic development. With the expansion of agricultural planting scale and the increase of planting years, crops have problems such as nutritional imbalance, pests and diseases. Farmers mainly prevent and control them by spraying pesticides and other chemical control methods. However, chemical pesticides have certain residual and toxicity, which have a certain impact on the environment. Excessive application can also cause soil compaction, acidification, secondary salinization and other problems, indirectly affecting the agricultural economy. Green control has attracted widespread attention due to its environmental protection and durability.

[0003] Plant growth promoting rhizobacteria (PGPR) is an environmentally friendly and green agricultural technology that protects soil and environment by promoting plant growth and preventing diseases and reducing the use of chemical pesticides. Rhizobacteria can compete with pathogens for nutrients in the soil, such as nitrogen, phosphorus, potassium, etc., and limit the growth and reproduction of pathogens. For example, patent CN 117126767 A discloses a potassium-solubilizing growth-promoting Enterobacter hollii and its bacterial agent and application. The rhizobacterium with potassium-solubilizing ability disclosed is HDC12, which can convert potassium in the soil that is difficult for plants to absorb and utilize into an available form, reduce the nutrients available to pathogens, and inhibit plant diseases caused by Fusarium to a certain extent. PGPR can secrete a variety of antibacterial substances, such as antibiotics, bacteriocins, volatile organic compounds (VOCs), etc., which can directly inhibit the growth of pathogens. For example, Bacillus subtilis can produce a variety of antibiotics and has antagonistic effects on a variety of plant pathogens. At the same time, PGPR can induce plants to produce systemic resistance (ISR), enhance the immunity of plants themselves, and enable plants to better resist diseases when infected by pathogens. For example, patent CN116286530A discloses a rhizosphere growth-promoting bacterium Bacillus Velez JB0319 with salt stress tolerance and its application. The disclosed Bacillus Velez JB0319 promotes the growth of lettuce under salt stress by improving the antioxidant enzyme activity in lettuce under salt stress, and activates the defense mechanism of plants. This type of microorganism creates good conditions for plant growth by improving the physical, chemical and biological properties of rhizosphere soil, and indirectly improves the disease resistance of plants. At the same time, this type of microorganism is used to make microbial agents, which is pollution-free to the environment. The microorganisms therein have functions such as nitrogen fixation, phosphorus solubilization and potassium solubilization, and can form a symbiotic relationship with the root system of crops, promote the absorption of nutrients by plants, and improve the fertilizer of soil. For example, patent CN 117070396 A discloses Pseudomonas and its application and a method for promoting plant growth. The fermentation liquid of Pseudomonas CCTCC NO: M 2023002 provided by the patent is used for root irrigation of plants, which can effectively increase the growth rate of plants, promote root development, and significantly increase the plant height, root length, stem girth and weight of plants. It has good nitrogen fixation and phosphorus solubilization effects, and can produce high iron carriers. Therefore, exploring this type of microbial resources and reducing environmental pollution are of great significance to the healthy development of the planting industry.

[0004] At present, in the research and development of plant rhizosphere growth-promoting bacteria, the screened and cultured microorganisms often only have two or three plant growth-promoting characteristics, such as phosphate solubilization, ammonium fixation, and iron carrier production, and only have certain effects on plant height, root length, and stem girth. It is not clear whether they have other growth-promoting functions. In addition, microorganisms often only have a control effect on a single pathogen and do not have broad-spectrum antibacterial properties. However, there are many pathogens in the soil, and a single control effect does not meet actual agricultural needs. At the same time, the process of using the microorganism to make microbial preparations is complicated and costly, making it difficult to promote and apply on a large scale. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus subtilis Gxunw-30 and application thereof.

[0006] The Bacillus subtilis Gxunw-30 provided by the present invention shows the ability to broadly inhibit plant pathogens such as Helicoverpa ryegrassii, Pseudococcus spp., Fusarium hechiense, Fusarium chlamydophorum, Pseudococcus graminearum, Botrytis cinerea, and Colletotrichum musa. At the same time, the strain has the performance of decomposing organic phosphorus, producing protease, ammonia, indole and fixing nitrogen, and has multiple growth-promoting properties that are beneficial to plants; and the strain contains genes encoding antibacterial substances, has the ability to synthesize iturin, surfactin, fengycin and bacilysin antibacterial peptides, and has potential antibacterial ability. The strain has the application prospect of preventing diseases and promoting growth, and provides a good biological control resource for preventing and controlling plant diseases.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first aspect of the present invention is to provide a Bacillus subtilis (Bacillus sp.) Gxunw-30, wherein the Bacillus subtilis Gxunw-30 is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a preservation number of GDMCC No: 65245 and a preservation date of October 12, 2024.

[0009] The second aspect of the present invention is to provide the fermentation broth of the above-mentioned Bacillus subtilis Gxunw-30.

[0010] The third aspect of the present invention is to provide a method for preparing the fermentation broth of the above-mentioned Bacillus subtilis Gxunw-30, comprising the following steps: inoculating a single colony of the above-mentioned Bacillus subtilis Gxunw-30 into LB liquid culture medium, and shaking the flask in a shaker at a temperature of 26 to 28°C and a rotation speed of 150 to 160 r / min for 22 to 24 hours to obtain the fermentation broth.

[0011] Furthermore, the concentration of Bacillus subtilis Gxunw-30 in the fermentation broth is 1×10 7 ~1×10 9 CFU / mL.

[0012] The fourth aspect of the present invention is to provide the use of the above-mentioned Bacillus subtilis Gxunw-30 and / or fermentation liquid in antagonizing plant pathogens, wherein the plant pathogens include Botryosphaeria dothidea, Fusarium hechiense, Cryphonectria parasitica, Lasiodiplodia theobromae, Fusariumpseudograminearum, Colletotrichum musae, Bipolaris sorokiniana and / or Fusarium chlamydosporum.

[0013] A fifth aspect of the present invention is to provide the use of the above-mentioned Bacillus subtilis Gxunw-30 and / or fermentation liquid in the preparation of preparations for preventing and controlling plant diseases caused by plant pathogens, wherein the plant pathogens include Botryosphaeria dothidea, Fusarium hechiense, Cryphonectria parasitica, Lasiodiplodia theobromae, Fusarium pseudograminearum, Colletotrichum musae, Bipolaris sorokiniana and / or Fusarium chlamydosporum.

[0014] Furthermore, the plant disease is plant wilt and / or root rot caused by Fusarium hechiense, Fusarium pseudograminearum and / or Fusarium chlamydosporum.

[0015] The sixth aspect of the present invention is to provide the use of the above-mentioned Bacillus subtilis Gxunw-30 and / or fermentation broth in the preparation of plant growth-promoting preparations.

[0016] The seventh aspect of the present invention is to provide a biocontrol agent comprising the above-mentioned Bacillus subtilis Gxunw-30 and / or the fermentation broth.

[0017] The eighth aspect of the present invention is to provide a method for preventing and controlling plant diseases, which comprises applying the above-mentioned biocontrol agent to plants.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0019] 1. The Bacillus subtilis Gxunw-30 provided by the present invention has the ability to broadly inhibit plant pathogens, and can antagonize plant pathogens such as Triticum aestivum, Pseudococcus spp., Fusarium hechiense, Fusarium chlamydophorum, chestnut blight pathogen, Pseudococcus graminearum, Botrytis cinerea, and banana anthracnose pathogens. The inhibition rate of pathogens is 59.52-87.2%, which can enhance the disease resistance of plants and provide a good biological control resource for preventing and controlling plant diseases.

[0020] 2. The Bacillus subtilis Gxunw-30 provided by the present invention can improve the efficiency of promoting plant growth and has multiple growth-promoting properties that are beneficial to plants, including the ability to hydrolyze organic phosphorus, produce proteases, produce ammonia, produce indoles and fix nitrogen, and has good application value for the development of plant growth-promoting preparations.

[0021] 3. The Bacillus subtilis Gxunw-30 provided by the present invention contains genes encoding antibacterial substances and has potential antibacterial ability. Bacillus subtilis Gxunw-30 has the ability to synthesize iturin, surfactin, fengycin and bacilysin antimicrobial peptides. Antimicrobial peptides play a vital role in disease resistance and have good application potential for the research and development of biocontrol agents.

[0022] 4. The Bacillus subtilis Gxunw-30 provided by the present invention can accelerate the growth rate of plants, make the root system of plants grow more developed, and eliminate the influence of Fusarium hechiense on plants, and has a preventive and therapeutic effect on diseases caused by the fungus.

[0023] 5. The microbial preparation culture process provided by the present invention is simple, low-cost, efficient and fast, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the appearance morphology of strain Gxunw-30 in Example 1

[0025] Figure 2 This is a diagram showing the identification of the organophosphorus-degrading performance of strain Gxunw-30 in Example 2.

[0026] Figure 3 This is a diagram for identifying the protease production performance of strain Gxunw-30 in Example 2.

[0027] Figure 4 This is a diagram for identifying the nitrogen fixation performance of strain Gxunw-30 in Example 2.

[0028] Figure 5 This is a diagram for identifying the ammonia production performance of strain Gxunw-30 in Example 2.

[0029] Figure 6 This is a diagram for identifying the indole production performance of strain Gxunw-30 in Example 2.

[0030] Figure 7 This is a diagram showing the broad-spectrum antibacterial effect of strain Gxunw-30 against plant pathogens in Example 3.

[0031] Figure 8 This is the phylogenetic tree diagram of strain Gxunw-30 constructed based on the 16SrDNA gene sequence in Example 4.

[0032] Fig. 9 This is the electrophoresis diagram of PCR detection of Gxunw-30 antimicrobial peptide related genes in Example 5.

[0033] Fig.10 This is a graph showing the results of a disease-resistant and growth-promoting potted plant experiment on strain Gxunw-30 in Example 6.

[0034] Fig.11 This is the root enzyme activity data of the potted experiment of strain Gxunw-30 in Example 7. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0036] In the present invention, Bacillus subtilis Gxunw-30 or simply referred to as strain Gxunw-30. The Bacillus subtilis provided by the present invention can be used as a liquid microbial agent. In the present invention, Bipolaris sorokiniana, Lasiodiplodia theobromae, Fusarium hechiense, Fusarium chlamydosporum, Cryphonectria parasitica, Fusarium pseudograminearum, Botryosphaeria dothidea, and Colletotrichum musae are all selected from the College of Ocean and Biotechnology, Guangxi University for Nationalities, and the Guangxi Key Laboratory of Polysaccharide Materials and Modification.

[0037] Example 1 Strain Isolation and Screening

[0038] Soil samples were collected from the rhizosphere of mango trees in Tianyang District, Baise City, Guangxi Zhuang Autonomous Region. 5 g of rhizosphere soil was weighed and placed in a round-bottom conical bottle. 45 mL of sterile water was added and placed in a constant temperature shaker at 35 °C and 180 r / min for 12 h. The soil suspension was diluted to 10 -3 , 10 -4 and 10 -5 , take 200 μL and evenly spread it on LB medium, repeat 3 times. After spreading, invert it in a constant temperature box at 30℃ and culture for 3 days. According to the colony characteristics of culturable microorganisms in solid medium, pick single colonies with different characteristics, separate and purify them by streaking on the plate until a single colony is obtained. The surface of the single colony of strain Gxunw-30 is white and sticky, convex, and moist inside ( Figure 1 ). Single colonies were collected with 30% glycerol and stored in a -80°C ultra-low temperature freezer.

[0039] Example 2 Screening of growth-promoting function of strains

[0040] The strain Gxunw-30 screened in Example 1 was activated by shake flask culture, and a screening medium for phosphate solubilization, protease production, ammonia production, indole production, and nitrogen fixation was prepared. A sterilized medium (solid plate or liquid) was prepared, and the medium was sterilized and inoculated. 1 μL of the bacterial solution was aspirated and spotted on the above-mentioned plate / sealed bottle, and placed at 28°C for culture. When a transparent circle or color reaction appeared, the experimental results were observed. Strains that were positive in various screening media were selected for further study.

[0041] Determination of phosphate solubilization ability: Inoculate the strain to be tested in the center of the Alexander Boroff medium, invert and culture at 28℃ for 5 days, and observe whether a transparent circle is produced. If so, it proves that it has the ability to solubilize phosphate. The calculation formula for the phosphate solubilization ability of the strain = diameter of the transparent circle of phosphate solubilization / colony diameter. The larger the ratio, the stronger the phosphate solubilization ability of the strain. Alexander Boroff medium: sucrose 5g, FeCl3 0.005g, CaCO3 0.1g, Na2HPO4 2g, MgSO4.7H2O 0.5g, (NH4)2SO4 1g, potassium feldspar powder 1g, agar 18g, distilled water 1L.

[0042] Protease production capacity determination: Inoculate the strain to be tested in the center of the milk agar medium, invert and culture at 28℃ for 2 days, repeat three times, and observe whether a transparent circle is produced. If it is produced, it proves that it has the ability to produce protease. The calculation formula for the strain's protease production capacity = plate transparent circle diameter / colony diameter. The larger the ratio, the stronger the protease production activity of the strain. Milk agar medium: 15g skim milk powder, 25g LB medium, 18g agar, 500mL distilled water, LB, and agar are sterilized, and then 500mL of sterilized and cooled skim milk powder is poured in.

[0043] Determination of ammonia production capacity: Inoculate the strain into 10 mL of peptone water liquid culture medium, culture at 28°C, 200 rpm for 36 hours, add 0.5 mL of Nessler reagent, if a yellow-brown precipitate appears, it indicates that it has the ability to produce NH3. Peptone water culture medium: 10 g of peptone, 10 g of sodium chloride, 1 L of distilled water, pH adjusted to 7.0-7.5.

[0044] Determination of indole production ability: Inoculate the bacteria into a test tube containing peptone liquid culture medium, place it in a 35℃ incubator for 1-2 days (can be extended to 4-5 days depending on the situation), slowly drip a few drops of Koch's reagent (about 0.5mL) along the wall of the tube, shake it gently while dripping and observing. If the color of the solution changes to rose red, it is positive (+), and if the color of the solution is not red, it is negative (-). Koch's reagent: Weigh 10g of p-dimethylaminobenzaldehyde and dissolve it in 150mL of 95% alcohol, then add 50mL of analytical grade concentrated hydrochloric acid.

[0045] Determination of nitrogen fixation ability: Inoculate the strain to be tested in the center of the Axubei nitrogen fixation medium, invert and culture at 28℃ for 3 days, repeat three times, and observe the growth of the strain. If the strain grows normally, it is judged to have the ability to fix nitrogen. Axubei nitrogen fixation medium: KH2PO4 0.2g, MgSO4.7H2O 0.2g, NaCl 0.2g, CaSO4 0.1g, CaCO3 5g, mannitol 10g, agar 18g, distilled water 1L, pH adjusted to 7.2.

[0046] The identification diagrams of the performance of degrading organic phosphorus, producing protease, fixing nitrogen, producing ammonia and producing indole of strain Gxunw-30 are shown in the figure below. Figures 2 to 6 Gxunw-30 showed a transparent circle in the center of the Alexander Borov medium, and the strain's ability to degrade organic phosphorus was 2.37 ( Figure 2 ); a transparent circle appeared in the center of the milk agar medium, and the strain's protease production capacity was 1.30 ( Figure 3 ); It can grow in the Axubei nitrogen-fixing medium, appears white and transparent, and has nitrogen-fixing properties ( Figure 4 ); the presence of a yellow-brown precipitation reaction in the peptone liquid medium indicates the ability to produce ammonia ( Figure 5 ); the pink color reaction in peptone liquid medium indicates the ability to produce indole ( Figure 6 ).

[0047] Example 3 Strain antagonism experiment

[0048] Take the strain Gxunw-30 purified in Example 1, and take eight plant pathogenic fungi, including Bipolarissorokiniana, Lasiodiplodia theobromae, Fusarium hechiense, Fusarium thickens, Cryphonectriaparasitica, Fusarium pseudograminearum, Botryosphaeria dothidea, and Colletotrichum musae, for flat plate confrontation, inoculate the pathogen cake (7.0 mm) in the center of the PDA plate culture medium, inoculate the test strain at 20 mm from the center of the plate, and use the PDA culture medium only inoculated with pathogens as a control, repeat three times, culture at 28°C for 5-7 days, observe the antibacterial effect of the strain, and calculate the antibacterial rate. The calculation formula of the antibacterial rate (%) = (the colony diameter of the control group - the colony diameter of the treatment group) / the colony diameter of the control group × 100%.

[0049] Figure 7The results show that strain Gxunw-30 has antagonistic effects on various plant pathogens in the plate antagonism experiment. The inhibition rate of strain Gxunw-30 on Triticum aestivum reached 66.6%, on Pseudo-cocoa Diplospora 74.6%, on Fusarium hechiense 79.3%, on Fusarium chlamydosporum 59.52%, on chestnut blight pathogen 78.6%, on Pseudo-grain Fusarium graminearum 71.7%, on Botrytis cinerea 87.2%, and on Colletotrichum muscifolium 67%.

[0050] Example 4 Molecular Biological Identification of Disease-Resistant and Growth-Promoting Bacteria

[0051] The strain Gxunw-30 in Example 2, which has the functions of solubilizing phosphate, producing protease, producing ammonia, producing indole, and fixing nitrogen, and has a broad-spectrum antibacterial ability against a variety of plant pathogens in Example 3, was preliminarily determined to be a strain with disease resistance and growth promotion potential, and molecular biological identification was performed on it.

[0052] Extract bacterial 16S rRNA gene fragments and identify species classification. The identification steps are as follows: Collect single colonies in the culture dish in a clean bench into a 1.5mL centrifuge tube, add 100μL 10% Chelex-100 solution, and oscillate to mix the bacterial solution. Boil the bacterial solution in boiling water for 30 minutes, centrifuge at 12000r / min for 10 minutes, take the supernatant as a DNA template, and use universal primers Eu-27F (5'-AGAGTTT-GATCCTGGCTCAG-3') and Eu-1492R (5'-GGT-TACCTTGTTACGACTT-3') to amplify bacterial 16S rRNA gene fragments. PCR reaction system (50μL): DNA template 2μL, 2×Taq Master Mix 25μL, upstream and downstream primers (10μmol / L) 1μL each, ddH2O 21μL. PCR program: 95℃ pre-denaturation for 4min, 95℃ denaturation for 1min, 55℃ annealing for 1min, 72℃ extension for 2min, 32 cycles, 72℃ full extension for 2min, and storage at 4℃. After detection by agarose gel electrophoresis, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rRNA gene sequencing results were compared by BLAST using the EzBioCloud database and NCBI.

[0053] The comparison results are as follows Figure 8 As shown, the results showed that strain Gxunw-30 and Bacillus subtilis were in the same branch, with a contribution value of 100%. Strain Gxunw-30 was identified as Bacillus subtilis and named Bacillus subtilis Gxunw-30.

[0054] The Bacillus subtilis Gxunw-30 is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), with a preservation number of GDMCC No: 65245 and a preservation date of October 12, 2024. The address is on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0055] Example 5 Detection of genes related to the synthesis of antimicrobial substances in strains

[0056] Take Gxunw-30 in Example 4 as the total DNA template, the steps are as follows: collect the single colony in the culture dish into a 1.5mL centrifuge tube on the clean bench, add 100μL 10% Chelex-100 solution, and oscillate to mix as a bacterial solution. Boil the bacterial solution in boiling water for 30min, centrifuge at 12000r / min for 10min, and take the supernatant as a DNA template. PCR amplification is performed on the genes encoded by the antibacterial substances iturin, bacillomycin, surfactin, fengycin, and bacilysin synthase, and the encoding genes are detected by 1% agarose gel electrophoresis (120V, 20min). If a bright band appears, it proves that the strain to be tested contains genes encoding antibacterial substances and has potential antibacterial ability. The amplification gene primer information is shown in Table 1, and the PCR reaction system is shown in Table 2.

[0057] Table 1 Amplification gene primer information

[0058]

[0059] Table 2 PCR reaction system

[0060]

[0061] The results of PCR electrophoresis for antimicrobial peptide-related genes are as follows Fig. 9 As shown by Fig. 9 It can be seen that Gxunw-30 has 6 bright bands in gel electrophoresis, namely ituA, ituD, srfAA, srfAC, fenD and bacA, indicating that Gxunw-30 may have the ability to synthesize iturin, surfactin, fengycin and bacilysin antimicrobial peptides, which play a vital role in disease resistance.

[0062] Example 6 Potted experiment of disease-resistant growth-promoting bacteria

[0063] Preparation of biocontrol strain fermentation broth: Pick a single colony on the solid culture medium plate of disease-resistant growth-promoting bacteria in Example 3, inoculate it into LB liquid culture medium with a liquid volume of 150 mL, and culture it in a shaker at a temperature of 28°C and a speed of 160 r / min for 24 hours. 8 CFU / mL, and the biocontrol strain fermentation liquid was made. Preparation of pathogen suspension: Pick hechiense Fusarium (Fusarium hechiense) on the PDA medium plate. The pathogen was isolated from the soil in the early stage and is corrosive to plant roots. Spraying the surface of mango fruit will accelerate the decay. Pick the mycelium and inoculate it in potato dextrose water (PDW). The liquid volume is 150mL. After shaking the flask at a temperature of 30℃ and a speed of 160r / min for 24 hours, the pathogen concentration is about 10 6 CFU / mL, and make a pathogen suspension.

[0064] Cucumber seedlings were planted in a greenhouse with a seedling medium. One cucumber seedling was planted in each seedling cup (specification 16cm×16cm). Inoculation was performed after 7 days of planting and reaching the 6-leaf stage. Control group: LB: sterile water = 3:147, 150 mL root irrigation; Treatment 1: 3 mL of pathogen suspension, pathogen: sterile water = 3:147, 150 mL root irrigation; Treatment 2: 3 mL of biocontrol strain, biocontrol bacteria: sterile water = 3:147, 150 mL root irrigation; Treatment 3: 50 mL of Fusarium pathogen solution with the same spore concentration as Treatment 2 was inoculated, and then 100 mL of biocontrol bacteria fermentation liquid with the same bacterial concentration as Treatment 1 was inoculated. Each treatment pot was repeated for 3 plants, and the roots were irrigated again after 3 days. The roots were irrigated for 7 consecutive times, and the relative temperature and relative humidity were kept consistent. The incidence rate and disease grade were investigated 21 days after inoculation, and the disease index and control effect were calculated.

[0065] 5-8 leaves of each potted plant were investigated, and each leaf was graded according to the percentage of the lesion area (S). The disease index and control effect were calculated according to the following formula:

[0066]

[0067] F=[(E e -E t ) / E e ]×100%(2)

[0068] E represents the disease index, n is the total number of leaves at each level, N is the total number of leaves investigated, S is the number of representatives at each level, S max is the highest representative value, F is the control effect, E is e is the disease index of the control group, E tis the disease index of the treatment group. The disease grading standard is to divide the percentage of leaf lesion area (S) into 6 levels. Level 0: S ≤ 10%; Level 3: 11% ≤ S ≤ 20%; Level 5: 21% ≤ S ≤ 30%; Level 7: 31% ≤ S ≤ 50%; Level 9: S > 50%.

[0069] Fig.10 The middle is a comparison of cucumber plants and roots in the treatment group and the control group after 21 days of root irrigation. Fig.10 It can be seen that after root irrigation with the fermentation liquid of strain Gxunw-30, the cucumber plants as a whole grew faster than those treated with root irrigation with LB medium only, with longer roots, thicker stems, more leaves, more developed cucumber root systems, and the effect of Fusarium hechiense on the plants could be eliminated.

[0070] Table 3 shows the growth index measurement results of cucumber seedlings in the control group and the experimental group.

[0071] Table 3 Growth of cucumber seedlings

[0072] Growth indicators Control group Processing 1 Process 2 Processing three Plant height (cm) 36.40 12.67 40.33 47.33 Number of leaves / plant 8 6 9 10 Stem thickness above ground (mm) 17.93 16.79 22.20 21.23 Root length (cm) 21.26 20.23 33.33 30.13 Fresh weight above ground (g) 10.56 5.02 13.66 15.24 Underground fresh weight 3.30 1.03 5.65 4.9 Disease index 0.30 0.41 0.34 0.37 Prevention Index - - 0.1150 0.2031

[0073] According to the data in Table 3, compared with the control group, the various indicators in treatment one (pathogen treatment group) were lower than those in the control group, and the disease index reached the highest. The height of cucumbers in treatment two (Gxunw-30 treatment group) and treatment three (Gxunw-30 and pathogen mixed treatment group) increased by 10.8% and 30%, respectively, the leaves increased by an average of 12.5% ​​and 25%, the aboveground stem diameter increased by 23.8% and 18.4%, the root length increased by 56.8% and 37%, and the aboveground fresh weight increased by 30.3% and 44.3%. After applying Gxunw-30, there was a certain control effect, with treatment two being 0.115 and treatment three being 0.2031.

[0074] Example 7 Investigation of Disease-Resistant Enzyme Activity in Cucumber Roots

[0075] The root tissues of the cucumber potted plants under each treatment in Example 6 were taken to investigate the anti-disease activity of the cucumber roots. According to the instructions of the enzyme activity kit (purchased from Beijing Solebow Technology Co., Ltd.), 0.1 g of cucumber root sample tissue was taken, and the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) in the roots were investigated by ultraviolet spectrophotometry.

[0076] The results of the investigation on the enzyme activity of the cucumber seedling roots treated with the fermentation liquid of strain Gxunw-30 are as follows Fig.11 As shown, Fig.11In the experiment, CK was the blank treatment, treatment one was the pathogen treatment, treatment two was the Gxunw-30 treatment group, and treatment three was the Gxunw-30 mixed with pathogen treatment. By detecting the four enzyme activities of cucumber roots, it was found that the activity of catalase would be increased after pathogen treatment, while the activities of the other three enzymes would be significantly lower than CK after pathogen treatment. The enzyme activities of the treatment with Gxunw-30 fermentation liquid alone (treatment two) were all higher than those of CK, indicating that Gxunw-30 fermentation liquid improved the disease resistance of cucumber roots. When Gxunw-30 was mixed with pathogens, Gxunw-30 could significantly inhibit the effect of pathogens on the activity of root enzymes, restore glutathione S-transferase and superoxide dismutase (SOD) to normal levels, and significantly increase peroxidase (POD).

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis Gxunw-30, characterized in that: The Bacillus subtilis Gxunw-30 is deposited in the Guangdong Provincial Microbiological Culture Collection Center, its preservation number is GDMCC No: 65245, and the preservation date is October 12, 2024.

2. A fermentation broth of Bacillus subtilis Gxunw-30 as claimed in claim 1.

3. A method for preparing a fermentation broth as claimed in claim 2, characterized in that: The method comprises the following steps: inoculating a single colony of the Bacillus subtilis Gxunw-30 described in claim 1 into an LB liquid culture medium, and culturing the culture in a shaking bottle at a temperature of 26-28° C. and a rotation speed of 150-160 r / min for 22-24 hours to obtain a fermentation liquid.

4. The method for preparing the fermentation liquid according to claim 3, characterized in that: The concentration of Bacillus subtilis Gxunw-30 in the fermentation broth is 1×10 7 ~1×10 9 CFU / mL.

5. Use of the Bacillus subtilis Gxunw-30 according to claim 1 and / or the fermentation liquid according to claim 2 in antagonizing plant pathogens, characterized in that: The plant pathogens include Botryosphaeria dothidea, Fusarium hechiense, Cryphonectria parasitica, Lasiodiplodia theobromae, Fusarium graminearum, Colletotrichum musae, Bipolaris sorokiniana and / or Fusarium chlamydosporum.

6. Use of the Bacillus subtilis Gxunw-30 according to claim 1 and / or the fermentation liquid according to claim 2 in preparing a preparation for preventing and treating plant diseases caused by plant pathogens, characterized in that: The plant pathogens include Botryosphaeria dothidea, Fusarium hechiense, Cryphonectria parasitica, Lasiodiplodia theobromae, Fusarium pseudograminearum, Colletotrichum musae, Bipolaris sorokiniana and / or Fusarium chlamydosporum.

7. The use according to claim 6, characterized in that: The plant diseases are plant wilt and / or root rot caused by Fusarium hechiense, Fusarium pseudograminearum and / or Fusarium chlamydosporum.

8. Use of the Bacillus subtilis Gxunw-30 according to claim 1 or the fermentation liquid according to claim 2 in preparing a plant growth-promoting preparation.

9. A biocontrol agent, characterized in that: Contains the Bacillus subtilis Gxunw-30 according to claim 1 and / or the fermentation broth according to claim 2.

10. A method for preventing and controlling plant diseases, characterized in that: The method comprises applying the biocontrol agent of claim 9 to plants.

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