Bacillus velezensis Gxunw-49 and application thereof

Abstract

The invention discloses bacillus velezensis Gxunw-49 and application thereof, and belongs to the technical field of agricultural microorganisms. The bacillus velezensis Gxunw-49 provided by the invention is preserved in Guangdong Microbial Culture Collection Center on October 12, 2024, and the preservation number of the bacillus velezensis Gxunw-49 is GDMCC No: 65246. The bacillus velezensis Gxunw-49 provided by the invention has the capability of inhibiting plant pathogens in a broad spectrum, and can be used for antagonizing plant pathogens such as hechiense fusarium, fusarium chlamydosporum, fusarium pseudograminearum, botryosphaeria dothidea and the like. The strain has the properties of producing siderophores, indole and protease, 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 bacillus velezensis Gxunw-49 disclosed by the invention has the effects of preventing diseases and promoting growth, provides a good biocontrol resource for prevention and treatment of soil-borne 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 Velez Gxunw-49 and applications thereof. Background Art

[0002] Soilborne diseases refer to pathogens living in the soil, such as bacteria, fungi, nematodes and viruses, which infect crops when conditions are right. Due to the dormant bodies, spores and eggs of the pathogens, they can often survive in the soil for a long time and are difficult to control and eliminate. Cucumber wilt is a typical soil-borne disease, mainly caused by Fusarium spp. in the soil. Infected cucumber seedlings may show symptoms such as slow growth and yellowing leaves. As the disease progresses, the leaves of cucumbers in the adult stage will gradually wither, until the entire plant dies. In cucumber-growing areas, once wilt breaks out on a large scale, the cucumber yield will drop significantly and the quality will also be seriously affected.

[0003] At present, the main methods for the prevention and control of cucumber wilt are chemical control, soil management and breeding of disease-resistant varieties. However, since Fusarium fungi are very easy to colonize in the soil, general chemical pesticides and control measures such as crop rotation and intercropping only have a short-term inhibitory effect. Excessive pesticides will also remain in the soil, destroying the microbial community structure, causing an increase in pathogens and a decrease in beneficial microorganisms. Therefore, biological control has attracted widespread attention due to its green environmental protection and durability. It is of great significance for studying the inhibition of pathogens such as Fusarium fungi and preventing the occurrence of soil-borne diseases. Biological control technology is a method of controlling diseases using natural biological resources. Studies have shown that exogenous addition of biocontrol strains such as Bacillus can inhibit the growth and infection of Fusarium fungal pathogens by competition, production of antibiotics or induction of plant defense responses. This type of microorganism is widely present in nature, is easy to isolate and culture, grows rapidly and has strong adaptability, has rich metabolites and has a broad spectrum of antibacterial activity, and is a microbial resource for making excellent anti-soil-borne disease bacterial preparations. There have been many reports on the green biological control function of Bacillus. For example, CN 114276177 A discloses a microbial organic fertilizer for preventing and controlling soil-borne diseases of cucumber, and its preparation method and application. It uses a composite bacterial agent of Pseudomonas aeruginosa ZM-1 and Bacillus amyloliquefaciens DS-1 and fermented with decomposed organic fertilizer to make a microbial organic fertilizer, which can effectively prevent and control soil-borne diseases of cucumber. And CN 115948270A discloses a strain antagonistic to soil-borne diseases, and its screening, identification method and application. It isolates, screens and identifies Bacillus siamensis and Bacillus velezensis from vinegar mash. Both microorganisms are rich in biosynthetic genes of antimicrobial peptides and can effectively prevent and control soil-borne wilt caused by Fusarium oxysporum.

[0004] However, the current stage of the exploration of soil microbial resources is not deep enough, and many microbial resources with disease resistance and growth promotion capabilities have not been well utilized. There are a variety of soil-borne pathogens in the soil, and bacterial preparations specifically made for a single pathogen often do not achieve the expected effect in practical applications. Only those with a wide range of antibacterial properties can effectively inhibit soil-borne diseases. At the same time, the current exploration of microorganisms only focuses on disease resistance, and often does not have other biological functions such as plant growth promotion. In addition, the process of using the microorganism to make microbial preparations is complex and costly, making it difficult to promote and apply on a large scale. Therefore, the exploration of antagonistic microorganisms that have a broad spectrum of inhibition of soil-borne pathogens, and have other biological functions such as plant growth promotion, as well as microorganisms that can be widely used in agricultural production, is a technical problem that urgently needs to be solved. Summary of the invention

[0005] The purpose of the invention is to provide a Bacillus Velez Gxunw-49 and application thereof.

[0006] The Bacillus Velezii Gxunw-49 provided by the present invention exhibits a broad spectrum of antibacterial ability against plant pathogens such as Hechiense Fusarium, Chlamydophorum, Pseudomonas graminearum, and Botrytis cinerea. At the same time, the strain has the ability to produce siderophores, indoles, and proteases, and has biological properties such as plant growth promotion; 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 an application prospect of disease prevention and growth promotion, and provides a good biological control resource for the prevention and control of soil-borne 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 sp. Gxunw-49, wherein the Bacillus sp. Gxunw-49 is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No: 65246 and a deposit date of October 12, 2024.

[0009] The second aspect of the present invention is to provide the fermentation broth of the above-mentioned Bacillus Velez subtilis Gxunw-49.

[0010] The third aspect of the present invention is to provide a method for preparing the fermentation broth of the above-mentioned Bacillus Velez Gxunw-49, comprising the following steps: inoculating a single colony of the above-mentioned Bacillus Velez Gxunw-49 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 Velezii Gxunw-49 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 Velez Gxunw-49 and / or fermentation broth in antagonizing plant pathogens, wherein the plant pathogens include Botrytis vinifera, Fusarium hechiense, Fusariumpseudograminearum and / or Fusarium chlamydosporum.

[0013] The fifth aspect of the present invention is to provide the use of the above-mentioned Bacillus Velez Gxunw-49 and / or fermentation liquid in the preparation of preparations for antagonizing soil-borne plant diseases caused by plant pathogens, wherein the plant pathogens include Botrytis vinifera, Fusarium hechiense, Fusariumpseudograminearum and / or Fusarium chlamydosporum.

[0014] Furthermore, the soil-borne plant diseases are plant wilt and / or root rot caused by Fusarium hechiense, Fusarium graminearum and / or Fusarium chlamydosporum.

[0015] The sixth aspect of the present invention is to provide the use of the above-mentioned Bacillus Velez Gxunw-49 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 Velezii Gxunw-49 and / or the fermentation broth.

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

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

[0019] 1. The Bacillus Velezii Gxunw-49 provided by the present invention has the ability to broadly inhibit plant pathogens, and can antagonize plant pathogens such as Hechiense Fusarium, Chlamydophora, Pseudomonas graminearum, and Botrytis cinerea, with an inhibition rate of 52.24-73.97% against the above pathogens, and can increase the activity of disease-resistant enzymes in plant roots and increase the disease resistance of plants. It provides a good biological control resource for the prevention and control of soil-borne diseases.

[0020] 2. The Bacillus Velez Gxunw-49 provided by the present invention can not only antagonize soil-borne pathogens in the soil, but also has biological properties such as plant growth promotion, including the production of siderophores, indoles, and proteases, and has good application value for the development of plant growth promotion preparations.

[0021] 3. The Bacillus Velez Gxunw-49 provided by the present invention contains genes encoding antibacterial substances and has potential antibacterial ability. Bacillus Velez Gxunw-49 has the ability to synthesize iturin, surfactin, fengycin and bacilysin antimicrobial peptides. Antimicrobial peptides play an important role in disease resistance and have good application potential for the research and development of biocontrol agents.

[0022] 4. The Bacillus Velezii Gxunw-49 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 Hechiense Fusarium 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-49 in Example 1.

[0025] Figure 2 This is a graph showing the performance of the iron carrier produced by strain Gxunw-49 in Example 2.

[0026] Figure 3 This is a diagram showing the protease production performance of strain Gxunw-49 in Example 2.

[0027] Figure 4 This is a diagram showing the indole production performance of strain Gxunw-49 in Example 2.

[0028] Figure 5 This is a diagram showing the broad-spectrum antibacterial effect of strain Gxunw-49 against plant pathogens in Example 3.

[0029] Figure 6 This is the electrophoresis diagram of PCR detection of antimicrobial peptide-related genes of strain Gxunw-49 in Example 4.

[0030] Figure 7 This is the phylogenetic diagram of strain Gxunw-49 constructed based on the 16S rRNA gene sequence in Example 5.

[0031] Figure 8 This is a graph showing the results of a potted plant experiment on the disease resistance and growth promotion of strain Gxunw-49 in Example 6.

[0032] Fig. 9 This is the enzyme activity diagram of the cucumber seedling roots of strain Gxunw-49 in Example 7. DETAILED DESCRIPTION

[0033] 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.

[0034] In the present invention, Bacillus sp. Gxunw-49 or simply referred to as strain Gxunw-49. The Bacillus sp. provided by the present invention can be used as a liquid bacterial agent. In the present invention, Fusarium hechiense, Fusarium chlamydosporum, Fusariumpseudograminearum, and Botrytis vinifera are all selected from the College of Ocean and Biotechnology, Guangxi University for Nationalities, and the Guangxi Key Laboratory of Polysaccharide Materials and Modification.

[0035] Example 1 Strain Isolation and Screening

[0036] Isolation from rhizosphere soil of mangrove plants in Guangxi Shankou Mangrove Ecological National Nature Reserve: 0.5 g of rhizosphere soil was placed in a round-bottom conical bottle, 50 mL of sterile water was added to make a soil suspension, and the suspension was placed in a constant temperature shaker at 30°C and 180 r / min for 12 h. The soil suspension was gradiently diluted to 10 -3 , 10 -4 and 10 -5 , take 100 μL and spread it evenly on LB medium, and repeat each gradient 3 times. After spreading, invert it in a constant temperature box at 30℃ and culture it for 3 days. According to the colony characteristics of culturable microorganisms in solid culture medium, pick colonies of different colors and properties, separate and purify them by streaking on the plate until single colonies are obtained. Collect single colonies with 30% glycerol and store them in a -80℃ ultra-low temperature refrigerator.

[0037] The strain finally screened out was numbered Gxunw-49, and its appearance is as follows Figure 1 As shown, Figure 1 It can be seen that the appearance of strain Gxunw-49 is white flakes with a moist, smooth and shiny surface and raised edges.

[0038] Example 2 Screening of growth-promoting function of strains

[0039] The strain Gxunw-49 screened in Example 1 was cultured in a shake flask, activated and expanded, and a screening medium for siderophore production, protease production, and indole production 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.

[0040] Determination of iron carrier production ability: Inoculate the strain to be tested in the center of the CAS detection medium, invert and culture at 28℃ for 4 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 iron carriers. The calculation formula for the ability of the strain to produce iron carriers = the diameter of the transparent circle on the plate / the diameter of the colony. The larger the ratio, the stronger the ability of the strain to produce iron carriers. CAS detection medium ingredients: Chrome azuro blue S (CAS) 60.5mg / L, hexadecyl trimethylammonium bromide (HDTMA) 72.9mg / L, hexahydrate ferric chloride 2.645mg / L, dihydrate sodium dihydrogen phosphate 295.25mg / L, dodecahydrate disodium hydrogen phosphate 1213.5mg / L, ammonium chloride 125mg / L, potassium dihydrogen phosphate 37.5mg / L, sodium chloride 62.5mg / L, agar 9g / L, pH value at 25℃ is 6.8±0.1.

[0041] The method for determining the protease production ability is as follows: the strain to be tested is inoculated in the center of the milk agar medium, and then inverted and cultured at 28°C for 2 days. The experiment needs to be repeated three times to observe whether the strain can produce a transparent circle. If the formation of a transparent circle is observed, it can be confirmed that the strain has the ability to produce protease. The calculation formula for the protease production ability is: plate transparent circle diameter / colony diameter. The larger the ratio, the stronger the protease production activity of the strain. The formula of milk agar medium is: 15g skim milk powder, 25g LB medium, 18g agar, and then diluted with 500mL distilled water. Finally, after sterilizing the LB medium and agar, pour them into 500mL of sterilized and cooled skim milk powder solution.

[0042] The method for determining the indole production ability is as follows: inoculate the bacteria into a test tube containing peptone liquid culture medium and place it in an incubator at 35°C for 1 to 2 days, which can be appropriately extended to 4 to 5 days depending on the specific strain. During the incubation process, slowly drip about 0.5 mL of Koch's reagent along the wall of the test tube, and gently shake the test tube to observe the reaction. If the color of the solution changes and becomes rose red, it is judged as positive (+); if the color of the solution does not turn red, it is judged as negative (-). The preparation method of Koch's reagent is: weigh 10g of p-dimethylaminobenzaldehyde, dissolve it in 150mL of 95% alcohol, and then add 50mL of analytical grade concentrated hydrochloric acid.

[0043] The siderophore production performance, protease production performance and indole production performance of strain Gxunw-49 are as follows: Figures 2 to 4 As shown, Gxunw-49 showed a transparent circle in the center of the CAS test medium, and the strain's ability to produce iron carriers was 1.62 ( Figure 2 ); a transparent circle appeared in the center of the milk agar medium, and the strain's protease production capacity was 2.06 ( Figure 3); the pink color reaction in the peptone liquid medium indicates the ability to produce indole ( Figure 4 ).

[0044] Example 3 Determination of the Antagonistic Bacterial Spectrum of the Strain

[0045] The strain Gxunw-49 with excellent growth-promoting function in Example 2 was selected, and a plate confrontation test was carried out on the following four plant pathogens: Fusarium hechiense, Fusarium chlamydosporum, Fusariumpseudograminearum and Botrytis vinifera. In the experiment, a pathogen cake with a diameter of 7.0 mm was inoculated in the center of the PDA plate culture medium, and the bacterial strain to be tested was inoculated 20 mm from the center of the plate. As a control group, a PDA culture medium inoculated only with pathogens was used. The experiment was repeated three times and maintained at a constant temperature of 28°C for 5-7 days. The antibacterial effect of different strains was observed and the antibacterial rate was then calculated. The calculation formula for the antibacterial rate (%) is: (control group colony diameter-treatment group colony diameter) / control group colony diameter × 100%.

[0046] The antagonistic and antibacterial effects of strain Gxunw-49 are as follows Figure 5 By calculation, the inhibition rate of Gxunw-49 against Fusarium chlamydophorum was 52.24%, against Fusarium Hechiense was 58.72%, against Botrytis cinerea was 73.97%, and against Fusarium pseudograminearum was 56.48%.

[0047] Example 4 Detection of genes related to the synthesis of antimicrobial substances in strains

[0048] Take the strain Gxunw-49 with excellent growth-promoting function in Example 2 and broad-spectrum antibacterial activity in Example 3 as the total DNA template, and the steps are as follows: collect the single colonies 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 of the genes encoding the antibacterial substances iturin, bacillomycin, surfactin, fengycin and bacilysin synthase, and detect the encoding genes 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 amplified gene primer information is shown in Table 1, and the PCR reaction system is shown in Table 2.

[0049] Table 1 Amplification gene primer information

[0050]

[0051] Table 2 PCR reaction system

[0052]

[0053] The results of PCR electrophoresis for antimicrobial peptide-related genes are as follows Figure 6 As shown by Figure 7 It can be seen that Gxunw-49 has five bright bands in gel electrophoresis, namely ituA, ituD, srfAA, srfAC, fenD, and bacA, indicating that Gxunw-49 may have the ability to synthesize iturin, surfactin, fengycin, and bacilysin antimicrobial peptides, which play a vital role in disease resistance.

[0054] Example 5 Species classification based on strain 16S rRNA gene fragments

[0055] Take the clean bench of Example 4 to collect the single colony in the culture dish into a 1.5mL centrifuge tube, 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, 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 the bacterial 16S rRNA gene fragment. 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°C pre-denaturation for 4 min; 94°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 2 min, 30 cycles, 32 cycles, 72°C extension for 2 min, and storage at 4°C. 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.

[0056] The comparison results are as follows Figure 7 As shown. Figure 7It can be seen that strain Gxunw-49 and Bacillus velezensis are in the same branch, with the maximum similarity reaching 100%. Strain Gxunw-49 was identified as Bacillus velezensis and named Bacillus sp. Gxunw-49.

[0057] The Bacillus sp. Gxunw-49 is deposited in the Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC No: 65246 and the deposit date October 12, 2024. The address is on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0058] Example 6 Potted experiment of disease-resistant growth-promoting bacteria

[0059] The preparation of the fermentation broth of the biocontrol strains was carried out by the following steps: a single colony was picked from the solid culture medium plate of the disease-resistant and growth-promoting bacteria in Example 3 and inoculated into 150 mL of LB liquid culture medium (formula: 1 g of tryptone, 0.5 g of yeast extract powder, 1 g of sodium chloride and 100 mL of sterile water). The culture was carried out in a shaker at 28°C and a speed of 160 r / min for 24 h. The bacterial cell concentration obtained after the culture was 10 8 CFU / mL, and the biocontrol strain fermentation broth was prepared.

[0060] The preparation steps of the pathogen suspension are as follows: Select the hyphae of Fusarium chlamydosporum from the PDA medium plate. The pathogen is isolated in the soil, has a putrefactive effect on plant roots, and can accelerate the decay of mango fruit. Pick a portion of the hyphae, inoculate it in 150mL of potato dextrose water (PDW), and culture it in a shaker at 30°C and a speed of 160r / min for 24h. The final pathogen concentration is about 10 6 CFU / mL, and make a pathogen suspension.

[0061] Cucumber seedlings were planted in a greenhouse using a seedling medium, with one cucumber seedling planted in each seedling cup (16cm×16cm in size). Seven days after planting, inoculation began when the seedlings reached the 6-leaf stage. Set up a control group: Mix LB with sterile water at a ratio of 3:147, and irrigate the roots with 150mL. Treatment 1: Take 3mL of pathogen suspension, mix it at a ratio of 3:147 between pathogen and sterile water, and irrigate the roots with 150mL. Treatment 2: Take 3mL of biocontrol strain, mix it at a ratio of 3:147 between biocontrol bacteria and sterile water, and irrigate the roots with 150mL. Treatment 3: Inoculate 50mL of Fusarium pathogen solution with the same concentration as treatment 1, and then inoculate 100mL of biocontrol bacteria fermentation solution with the same bacterial concentration as treatment 2. Repeat 3 plants for each treatment, irrigate the roots again 3 days after treatment, and irrigate the roots 7 times in a row to ensure that the relative temperature and humidity of the environment remain consistent. 21 days after vaccination, the incidence and disease grade were investigated, and the disease index and prevention and treatment effect were calculated.

[0062] 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:

[0063]

[0064] F=[(E e -E t ) / E e ]×100%(2)

[0065] In the above formula, 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, and S max is the highest representative value, F is the control effect, E is e is the disease index of the control group, E t is 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%.

[0066] Comparison of cucumber plants and roots in the treatment group and control group after 21 days of root irrigation Figure 8 As shown in Figure 8, it can be seen that after root irrigation with the fermentation liquid of strain Gxunw-49, the cucumber plants as a whole grew faster than those treated with root irrigation with LB medium alone, with longer roots, thicker stems, more leaves, more developed cucumber root systems, and the effect of Fusarium chlamydosporum on the plants could be eliminated.

[0067] The growth index measurement results of cucumber seedlings in the control group and the experimental group are shown in Table 3:

[0068] Table 3 Growth of cucumber seedlings

[0069]

[0070]

[0071] According to the data in Table 3, compared with the control group, the various indicators in treatment 1 (pathogen treatment group) were lower than those in the control group, and the disease index reached the highest. The height of cucumbers in treatment 2 (Gxunw-49 treatment group) and treatment 3 (Gxunw-49 and pathogen mixed treatment group) increased by 12.63% and 31.86%, respectively, the leaves increased by an average of 12.5% ​​and 33.25%, the aboveground stem diameter increased by 10.65% and 18.9%, the root length increased by 19.8% and 37.48%, and the aboveground fresh weight increased by 26.6% and 5%. After applying gxunw-29, there was a certain control effect, with treatment 2 being 0.090 and treatment 3 being 0.261.

[0072] Example 7 Investigation of Disease-Resistant Enzyme Activity in Cucumber Roots

[0073] 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.

[0074] The results of the investigation on the enzyme activity of the cucumber seedling roots treated with the fermentation liquid of strain Gxunw-49 are as follows Fig. 9 As shown, Fig. 9 In the experiment, CK was the blank treatment, treatment one was the pathogen treatment, treatment two was the Gxunw-49 treatment group, and treatment three was the Gxunw-49 mixed with pathogens. By testing the four enzyme activities of cucumber roots, it was found that after the pathogen treatment, except for the increase in catalase activity relative to CK, the activities of the other three enzymes were significantly lower than those of CK. The enzyme activities of the treatment with Gxunw-49 fermentation liquid alone were higher than those of CK, indicating that the Gxunw-49 fermentation liquid improved the disease resistance of cucumber roots. When Gxunw-49 was mixed with pathogens, Gxunw-49 could significantly inhibit the effect of pathogens on the enzyme activity of the roots and restore it to the level before infection.

[0075] 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 velezensis Gxunw-49, characterized in that: The Bacillus Velez Gxunw-49 is deposited in the Guangdong Provincial Microbiological Culture Collection Center, its preservation number is GDMCC No: 65246, and the preservation date is October 12, 2024.

2. A fermentation broth of Bacillus Velez subtilis Gxunw-49 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 Velezii Gxunw-49 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 Velez Gxunw-49 in the fermentation broth was 1×10 7 ~1×10 9 CFU / mL.

5. Use of the Bacillus Velez Gxunw-49 according to claim 1 and / or the fermentation liquid according to claim 2 in antagonizing plant pathogens, characterized in that: The plant pathogens include Botrytis vinifera, Fusarium hechiense, Fusarium graminearum and / or Fusarium chlamydosporum.

6. Use of the Bacillus Velez Gxunw-49 according to claim 1 and / or the fermentation liquid according to claim 2 in preparing a preparation for antagonizing soil-borne plant diseases caused by plant pathogens, characterized in that: The plant pathogens include Botrytis vinifera, Fusarium hechiense, Fusarium pseudograminearum and / or Fusarium chlamydosporum.

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

8. Use of the Bacillus Velez Gxunw-49 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: It comprises the Bacillus Velez Gxunw-49 according to claim 1 and / or the fermentation broth according to claim 2.

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

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