Banana endophytic bacillus velezensis JDB15 and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to banana endophytic bacillus velezensis JDB15 and application thereof. The novel endophytic bacillus velezensis JDB15 is separated from banana plants, has the characteristics of efficient inhibition of Foc4, growth promotion of bananas and broad-spectrum antagonism of various pathogenic bacteria, can inhibit the morbidity of corn southern leaf blight, tomato fusarium wilt, tobacco gray mold and peach anthracnose, has the effects of inhibiting the morbidity of various pathogenic fungi, and has the advantages of high efficiency, low cost and the like. And a good broad-spectrum effect is achieved. Meanwhile, the strain can kill nematodes, has wide biocontrol bacterium activity, can be developed deeply subsequently, and provides a sustainable and green solution for prevention and treatment of banana wilt and other diseases and insect pests.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to an endophytic banana Bacillus Velez subtilis JDB15 and an application thereof. Background Art

[0002] banana( Musa As one of the most important economic crops in the world, banana wilt (Bana fusarium wilt) is widely planted in about 130 countries and regions around the world, with an annual output of more than 100 million tons. It is the mainstay of the agricultural economy in tropical and subtropical regions. Fusarium The spread of Fusarium oxysporum f. Fusarium oxysporum f. sp. cubense , Foc ) is a typical soil-borne fungal disease that causes vascular necrosis. It was first discovered in Hawaii in 1904 and quickly spread to banana-producing areas around the world. Once the disease breaks out, the incidence rate can reach 10%-40%, with more than 90% of the land affected being severely affected, leading to a significant reduction in banana yields or even complete abandonment. It is highly destructive and difficult to eradicate, causing huge economic losses to banana farmers around the world. Even more serious is that the banana wilt pathogen race 4 ( Foc 4) It can infect a variety of cultivated varieties including Cavendish, which accounts for more than 95% of the global banana trade, which means that the disease threat has reached the core of the industry. At present, the prevention and control of banana wilt mainly relies on chemical pesticides, but long-term use has disadvantages such as drug residues, poor environmental compatibility, and low prevention and control efficiency. Biological control technology with microbial preparations as the core is regarded as an ideal solution to replace chemical pesticides. Among them, plant endophytes have the advantages of strong environmental affinity, high colonization stability, and diverse metabolic functions due to co-evolution with the host. However, some strains are isolated from non-banana hosts (such as apples and vegetables), and their colonization efficiency and Foc 4 is insufficiently effective, and live microbial preparations are susceptible to storage conditions, resulting in a short shelf life that restricts their industrial application. Furthermore, most strains focus on disease prevention and control, while their effects on promoting banana growth and regulating nutrient metabolism have not been fully explored. Therefore, given these challenges, the development of strains antagonistic to banana Fusarium wilt is of great significance to the development of the banana industry. Summary of the Invention

[0003] The present invention provides a banana endophytic Bacillus Velez JDB15 and its application, which has both Foc 4. Highly effective inhibition, banana growth promotion and multi-pathogen broad-spectrum antagonism.

[0004] The technical solution of the present invention is achieved as follows: The first aspect of the present invention is a banana endophytic Bacillus Velez JDB15, named Bacillus velezensis , registered and deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 66431.

[0005] The second aspect of the present invention is to provide the fermentation broth or the filtrate of the fermentation broth of the banana endophytic Bacillus Velez subtilis JDB15 as described in the first aspect of the present invention.

[0006] The third aspect of the present invention is to provide a preparation containing the banana endophytic Bacillus Velez subtilis JDB15 described in the first aspect of the present invention or the fermentation broth described in the second aspect of the present invention.

[0007] The fourth aspect of the present invention provides the use of the banana endophytic Bacillus Velez JDB15 described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in preparing a preparation antagonistic to banana Fusarium wilt, gray mold, Fusarium graminearum, rice blast, sclerotinia sclerotiorum, Rhizoctonia solani, rapeseed wilt, peanut white rot, Fusarium fusariococcus, peach anthracnose, peach brown rot, Fusarium oxysporum tomato-specific type, Alternaria alternata, corn leaf blight, bacterial leaf blight and bacterial leaf streak.

[0008] The fifth aspect of the present invention provides the use of the banana endophytic Bacillus Velez JDB15 described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in the preparation of a preparation for preventing and controlling diseases caused by banana Fusarium wilt, gray mold, Fusarium graminearum, rice blast, sclerotinia sclerotiorum, Rhizoctonia solani, rapeseed wilt, peanut white rot, Fusarium fusariococcus, peach anthracnose, peach brown rot, Fusarium oxysporum tomato transformed form, Alternaria alternata, corn leaf blight, bacterial leaf blight and / or bacterial leaf streak, or for preventing and controlling nematodes.

[0009] The sixth aspect of the present invention provides the use of the banana endophytic Bacillus Velez JDB15 described in the first aspect of the present invention, or the fermentation broth or filtrate of the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in the preparation of a preparation for improving the resistance of banana plants to banana wilt pathogen, inhibiting the mycelial growth of banana wilt pathogen, and inhibiting the spore germination of banana wilt pathogen.

[0010] The seventh aspect of the present invention provides the use of the banana endophytic Bacillus Velez subtilis JDB15 described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in promoting the growth of banana plants.

[0011] Furthermore, the banana endophytic Bacillus Velez subtilis JDB15 described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention can increase the stem diameter, plant height, leaf area, plant fresh weight, plant dry weight and / or root number of banana plants.

[0012] The eighth aspect of the present invention provides the use of the banana endophytic Bacillus Velez JDB15 described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in the preparation of a preparation that secretes protease, cellulase, amylase, pectinase, auxin, nitrogen fixation, potassium solubilization, organophosphorus solubilization and / or chitinase activity.

[0013] The ninth aspect of the present invention provides the use of the banana endophytic Bacillus Velez subtilis JDB15 described in the first aspect of the present invention, or the fermentation liquid described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention in preparing a preparation for improving the stress resistance of banana plants.

[0014] Furthermore, the banana endophytic Bacillus Velez JDB15 described in the first aspect of the present invention, or the fermentation broth described in the second aspect of the present invention, or the preparation described in the third aspect of the present invention can increase the peroxidase activity and / or catalase content in normal banana plants and banana plants infected with banana wilt pathogen.

[0015] Beneficial effects of the present invention: The present invention isolates a novel endophytic Bacillus Velezii JDB15 from banana plants. Foc 4. Highly efficient inhibition, banana growth promotion and broad-spectrum antagonism against multiple pathogens. It can inhibit the incidence of rice blast, corn leaf spot, tomato wilt, tobacco gray mold, and peach anthracnose. It has the ability to inhibit the incidence of multiple pathogenic fungi and has a good broad-spectrum effect. At the same time, the strain can also kill nematodes and has a wide range of biocontrol activity. It can be further developed in the future to provide sustainable and green solutions for the prevention and control of banana wilt and other diseases and pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 For strain JDB15 Foc 4. Results of mycelial antagonism experiment.

[0018] Figure 2 This is the 16s amplification result of strain JDB15.

[0019] Figure 3 Phylogenetic tree of strain JDB15.

[0020] Figure 4 These are the culture characteristics of strain JDB15 on seven culture media.

[0021] Figure 5 This is the Gram staining result of strain JDB15.

[0022] Figure 6 These are the results of physiological activity determination of strain JDB15.

[0023] Figure 7 These are the results of the NaCl tolerance experiment of strain JDB15.

[0024] Figure 8 These are the results of the broad-spectrum antifungal activity test of strain JDB15.

[0025] Figure 9 These are the results of the antibacterial activity test of strain JDB15 against PX099 and XOC.

[0026] Figure 10 The filtrate of the fermentation broth of strain JDB15 Foc 4. The results of the inhibitory activity assay.

[0027] Figure 11 The filtrate of the fermentation broth of strain JDB15 Foc 4. The results of mycelial inhibition activity assay.

[0028] Figure 12 The filtrate of the fermentation broth of strain JDB15 Foc 4. Results of spore germination inhibition activity assay.

[0029] Figure 13 This is the control effect of the fermentation liquid of strain JDB15 on banana wilt.

[0030] Figure 14 This is the growth-promoting effect of the fermentation liquid of strain JDB15 on banana plants.

[0031] Figure 15 This is the effect of the fermentation liquid of strain JDB15 on the growth of banana plants.

[0032] Figure 16 These are the results of enzyme activity detection of banana seedlings using the fermentation liquid of strain JDB15.

[0033] Figure 17The control effect of the fermentation liquid of strain JDB15 on barley blast.

[0034] Figure 18 This is the control effect of the fermentation liquid of strain JDB15 on corn leaf blight.

[0035] Figure 19 This is the control effect of the fermentation liquid of strain JDB15 on nematodes.

[0036] Figure 20 This is the control effect of the fermentation liquid of strain JDB15 on tomato wilt.

[0037] Figure 21 This is the control effect of the fermentation liquid of strain JDB15 on tobacco gray mold.

[0038] Figure 22 The control effect of the fermentation liquid of strain JDB15 on peach anthracnose.

[0039] The banana endophytic Bacillus Velez subtilis JDB15 of the present invention is named: Bacillus velezensis It is registered and deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No: 66431 and the deposit date is May 28, 2025. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0041] Example 1

[0042] 1. Isolation of Endophytes and Determination of Antifungal Activity First, some endophytic strains were isolated from banana roots using the relevant endophyte isolation procedures.Banana root samples were collected from the greenhouse of Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, Hubei Province (Longitude: 114.3E Latitude: 30.4N).

[0043] Endophytic bacteria from bananas were isolated using the tissue dilution method. Banana plants with good growth and no lesions were selected for isolation. Prior to the experiment, forceps, blades, filter paper, a coating rod, a 2ml centrifuge tube, and small steel balls were sterilized with high-temperature steam. A solution of 50% glycerol, 75% ethanol, and 1% hypochlorous acid was prepared for use. Surface disinfection: First, the banana roots were rinsed with tap water and cut into small pieces. Surface disinfection was performed in a sterile operating hood: first, rinsed with running water, allowed to dry, and then transferred to a clean bench. Rinse with 75% ethanol for 1 minute, rinsed once with sterile water, then rinsed with 1% sodium hypochlorite for 30 seconds, and rinsed three times with sterile water. Surface moisture was then blotted dry with filter paper. 100 µL of the supernatant from the final sterile water wash was spread onto PDA and LB medium. Three blank controls were incubated at 28°C and 30°C for 1-5 days. No bacterial growth was observed, indicating that the banana root surface was thoroughly disinfected. Set up a concentration gradient: Place 0.1 g of banana root tissue in a 2 ml EP tube with steel balls, shake it thoroughly with a vortex shaker, dilute the supernatant with ultrapure water, and set up 4 concentration gradients of 10 -1 -10 -4 . Dilution plate culture: Repeat each gradient three times, take 0.1 ml and spread it on the LB plate, place it in a 30℃ incubator and culture it for 1-2 days, and observe the number of colonies during this period. Purification culture: When colonies of various morphologies grow on the plate, use a sterilized toothpick to select strains with obviously different morphologies and activate them on LB. Preservation of endophytic bacteria: Pick a single colony and place it in an EP tube containing LB liquid culture medium, shake it at 28℃, 180 r / min for 12 hours, add the bacterial solution and 50% glycerol in a 1:1 ratio, mix well and freeze at -70℃ (can be stored at -20℃ for half a year) for the next experiment.

[0044] Then the isolated strains were tested for Foc 4. Inhibitory ability of mycelial growth. Antagonistic test was performed by plate antagonism test and counter-culture to screen Foc 4. Bacteria: Pour 10 mL of PDA medium into a 6 cm culture dish and set aside. Take fresh purified bacterial strains and draw lines perpendicular to the diameter at 1.5 cm from the center on both sides of the poured PDA plate. Incubate at 30°C for 1 day. After the colonies grow, use a 5 mm diameter punch to punch fresh Foc Punch a hole at the edge of the hyphae of the 4 strains and inoculate the hyphae block in the center of the PDA plate. Incubate at 25℃ and observe the confrontation. Sterile water was used as a control. Foc 4. When the strains are fully grown on the PDA plate, observe whether there is an antagonistic zone. If there is antagonism, measure the width of the antagonistic zone and identify it, and select the strongly antagonistic strain for the next test.

[0045] During the experiment, it was found that the endophyte numbered JDB15 Foc 4 Mycelial growth showed obvious antagonism ( Figure 1 ), the inhibition rate was 80.34%.

[0046] 2. Analysis of growth characteristics of JDB15 Genomic DNA from strain JDB15 was extracted using the Omega Becterial DNA Kit. PCR amplification of the target fragment was performed using the bacterial 16S rDNA gene primers: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGCTACCTTGTTACGACTT-3'. The PCR reaction system was as follows: PCR amplification conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 30 sec, 32 cycles; 72℃ extension for 8 min, 12℃ stop. A sequence of about 1500 bp was obtained ( Figure 2 ). The 16S RNA sequence of strain JDB15 was compared with that of Bacillus velezinis in the GenBank database and was found to be most similar to that of Bacillus velezinis.

[0047] By comparing with the local database, the 19 closest strains at the species level were selected based on 31 housekeeping genes ( dnaG , frr , infC , nusA , pgk , pyrG , rplA , rplB , rplC , rplD , rplE , rplF , rplK , rplL , rplM , rplN , rplP , rplS , rplT , rpmA , rpoB , rpsB , rpsC , rpsE , rpsI , rpsJ , rpsK , rpsM , rpsS , smpB , tsf ), and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA 6.0 software ( Whole-genome sequencing by Meiji Biotechnology ) ( Figure 3 ).

[0048] The above results confirm that JDB15 is Bacillus velezensis. To further examine its morphological changes, scanning electron microscopy revealed that the cells ranged from 1.20 to 1.80 μm in length and 0.5 to 0.75 μm in width. The cells had a smooth surface and were accompanied by filaments. Bacillus velezensis can secrete extracellular polymers (such as polysaccharides, proteins, or DNA) that may form filamentous networks, connecting multiple cells or encapsulating bacterial colonies to form biofilms. It can also secrete antimicrobial substances (such as lipopeptides) and extracellular polymers (EPS) that form filamentous structures that encapsulate the cells.

[0049] The strain JDB15 was cultured on seven different media, including LB, PDA, TSA, NA, R2A, Gause's No. 1, King B medium ( Figure 4 ).

[0050] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar.

[0051] PDA medium: 200 g potatoes, 20 g glucose, 15 g agar.

[0052] TSA medium: 15 g tryptone, 5 g soytone, 5 g sodium chloride, 15 g agar.

[0053] NA medium: peptone 10 g, beef powder 3 g, sodium chloride 5 g, agar 15 g.

[0054] R2A medium: yeast extract powder 0.5 g, peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, soluble starch 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium pyruvate 0.3 g, magnesium sulfate 0.024 g, agar 15 g.

[0055] Gause's medium No. 1: soluble starch 20g, potassium nitrate 1g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, sodium chloride 0.5g, ferrous sulfate 0.01g, agar 15g.

[0056] King B medium: 20.0g peptone, 1.5g potassium hydrogen phosphate, 1.5g sulfuric acid heptahydrate, 15g agar. On PDA, LB, TSA, NA and King B medium, the colonies appear light yellow, on R2A medium they appear beige, and on Gause's No. 1 medium they appear white. A pungent odor and biofilm will be produced on LB medium, the diameter of the raised colonies is the largest, and a large amount of bacterial metabolites are also observed, appearing as mucus-like and translucent water-soaked products. A special odor can be smelled on all culture media, and multiple notches on the edges of the colonies, irregular growth, rough appearance, opacity and wrinkled surface are observed, which can be used as identification criteria for Bacillus. Staining by Gram staining method found that the bacterial cells of JDB15 appeared purple, and it was identified as a Gram-positive strain ( Figure 5 ). Therefore, based on the above, strain JDB15 was identified as Bacillus velezensis and named: Bacillus velezensis It is registered and deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No: 66431 and the deposit date is May 28, 2025. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0057] 3. Physiological, biochemical and growth-promoting properties of JDB15 Cellulose Congo red medium: 1 g potassium dihydrogen phosphate, 2 g ammonium sulfate, 0.5 g magnesium sulfate heptahydrate, 2 g CMC-Na, 0.5 g sodium chloride, 0.4 g Congo red, 15 g agar, and 1000 mL distilled water.

[0058] Amylase detection medium: 2 g soluble starch, 10 g beef extract, 10 g glucose, 7 g peptone, 18 g agar, and 1000 mL deionized water.

[0059] Pectinase detection medium: Glucose: 20g, pectin 5g, agar powder 15g, potato extract 200g. Cut the potatoes into pieces and boil them for 30 minutes. Filter the juice and add water to 1L.

[0060] Protease assay medium: 5 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar, 10 g skim milk powder, 1 L distilled water.

[0061] Siderophore production detection medium: 60.5 mg chrome azurol-S, 72.9 mg hexadecyltrimethylammonium bromide, 2.645 mg ferric chloride hexahydrate, 295.25 mg sodium dihydrogen phosphate dihydrate, 1213.5 mg disodium hydrogen phosphate dodecahydrate, 125 mg ammonium chloride, 37.5 mg potassium dihydrogen phosphate, 62.5 mg sodium chloride, 15 g agar, pH 6.8 ± 0.1, 1000 mL distilled water.

[0062] Ashby solid medium (nitrogen-fixing): 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5 g calcium carbonate, 10 g mannitol, 0.1 g calcium sulfate dihydrate, 15 g agar, 1 L pure water, pH 7.0-7.5.

[0063] Pikovskaya medium (inorganic phosphate): glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate heptahydrate 0.3 g, manganese sulfate monohydrate 0.03 g, potassium chloride 0.3 g, ferrous sulfate heptahydrate 0.03 g, tricalcium phosphate 5.0 g, yeast extract 0.5 g (optional), agar 15.0 g, purified water 1 L, pH 7.0 ± 0.2.

[0064] Montana organophosphate medium: glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate heptahydrate 0.3 g, manganese sulfate monohydrate 0.03 g, potassium chloride 0.3 g, ferrous sulfate heptahydrate 0.03 g, lecithin 2.0 g, yeast extract 0.5 g (optional), agar 15.0 g, purified water 1 L, pH 7.0 ± 0.2g Chitinase detection medium: yeast powder 5g, peptone 5g, NaCl 5g, chitin 10g, agar 15-20g, distilled water 1000ml, pH 7.0-7.2.

[0065] Dextran agar medium: 4.0 g of Poria powder, 17.9 g of disodium hydrogen phosphate, 6.8 g of potassium dihydrogen phosphate, 6.7 g of yeast powder, 0.06 g of aniline blue, 15 g of agar, dilute to 1000 mL with distilled water, pH 7.2.

[0066] Alexandrov medium: 2 g disodium hydrogen phosphate, 1 g potassium feldspar powder, 0.5 g magnesium sulfate heptahydrate, 0.1 g calcium carbonate, 0.05 g ferric chloride, 5 g sucrose, 15 g agar, and 1000 mL pure water.

[0067] (1) Starch hydrolysis test: JDB15 bacteria were inoculated into a starch-containing culture medium and cultured in a 28°C incubator for 3 days. The colonies were stained with iodine solution. If there was a clear transparent circle around the colony, it was a positive bacterium.

[0068] (2) Cellulase assay method: strain JDB15 was inoculated onto a CMC plate according to the above method. After being placed in a 28°C incubator for 3 days, an appropriate amount of Congo red solution (1 mg / mL) was poured onto the plate and stained for 15 minutes. The plate was then rinsed several times with sodium chloride solution (1 mol / L). Whether a yellow halo appeared around the colony was observed. If so, it indicated that the strain could produce cellulase. The diameter of the halo was measured and recorded.

[0069] (3) Pectinase assay method: Inoculate strain JDB15 onto a pectin culture medium plate according to the above method. After placing it in a 28°C incubator for 3 days, stain the colonies with iodine solution. If there is a clear transparent circle around the colony, it is a positive bacterium.

[0070] (4) Protease assay: Place a sterile filter paper disc in the center of a skim milk agar plate. Take 10 μL of the test strain cultured in LB for 24 h and slowly inoculate it onto the filter paper disc. Place the plate in a 28°C incubator for 3 days. If a clear zone forms around the colony, the strain can produce protease. Measure and record the diameter of the clear zone.

[0071] (5) Iron carrier determination method: CAS plates were used to detect the production of iron carriers. The strain was inoculated in the center of the CAS plate and cultured at 28°C for 3 to 5 days. The colony was observed to see whether an orange halo was formed around it. If so, it indicated that the strain could secrete iron carriers. The diameter of the halo was measured and recorded.

[0072] (6) Auxin IAA assay: The IAA production was quantitatively determined using a colorimetric method. The strain was inoculated into 4 mL of LB medium containing 40 mg of tryptophan. LB medium without the strain was used as a negative control. After incubation at 28°C for 48 h, 1.5 mL of the culture medium was taken and centrifuged at 12100 rpm for 10 min. 0.5 mL of the supernatant was mixed with 1 mL of Salkowski reagent, placed in a 1.5 mL tube, and incubated in the dark at 25°C for 30 min. The production of IAA was determined by color change.

[0073] (7) Determination of inorganic phosphorus: JDB15 was activated in LB solid medium and cultured at 28°C for 48 h. Then, a sterile toothpick was used to pick up the colonies and inoculate them in Pikovskaya medium in a clean bench.

[0074] (8) Method for determination of organophosphorus: JDB15 was activated using LB solid medium and cultured at 28°C for 48 h. Then, a sterile toothpick was used to pick up the colonies and inoculate them on Montgena organophosphorus medium in a clean bench.

[0075] (9) Nitrogen fixation determination method: JDB15 was activated using LB solid medium and cultured at 28°C for 48 h. Then, in a clean bench, a sterile toothpick was used to pick up the colony and inoculate it on Ashby solid medium.

[0076] (10) Potassium dissolution assay: JDB15 was activated in LB solid medium and cultured at 28°C for 48 h. Then, a sterile toothpick was used to pick up the colonies and inoculate them in Alexandrov medium in a clean bench.

[0077] (11) Chitinase detection method: The bacteria were first streaked on an LB plate and cultured at 28°C for 48 h. The plate was coated with about 10 ml of sterile water to prepare a bacterial suspension. Then 1 μl of the suspension was spotted on a colloidal chitin plate and cultured until a transparent circle appeared.

[0078] (12) Determination method of dextranase detection medium: Use LB solid medium to activate JDB15, culture at 28°C for 48 hours, and then use a sterile toothpick to pick up the colony and inoculate it on dextran agar medium in an ultra-clean workbench.

[0079] The results of the test are shown in Figure 6 As shown in Table 1, strain JDB15 has a strong ability to secrete proteases, with a clear zone diameter of 25.6 mm. It also secretes cellulases, with a halo diameter of 40.5 mm. It also has strong amylase (30.4 mm in diameter) and pectinase (35.5 mm in diameter) activities, and is capable of producing IAA. It lacks the ability to produce siderophores. It also has the ability to fix nitrogen and degrade potassium, and it produces chitinase and degrade organic phosphorus, but lacks the ability to degrade inorganic phosphorus or produce β1,3-glucanase.

[0080] Table 1 Physiological and biochemical test results of strain JDB15 Physiology and Biochemistry result Growth promoting properties result amylase + IAA + Cellulase + Solution of inorganic phosphorus - Pectinase + Organic phosphorus solution + Protease + Nitrogen fixation + Siderophore-producing - Potassium + Chitinase + β1,3-glucanase - (13) NaCl tolerance experiment: In order to explore the tolerance of the strain to NaCl, LB culture medium containing different NaCl concentrations (0%, 2%, 4%, 6%, 8%, 10%) was prepared to ensure that other nutritional conditions were consistent. The strains were inoculated into the culture medium and placed at 28°C for 3 days. After the culture was completed, the growth of the strains was observed and their growth ability at different NaCl concentrations was recorded to determine the upper and lower limits of the strain's tolerance to NaCl. The results are shown in Figure 2. Figure 7 and as shown in Table 2.

[0081] Table 2 Results of NaCl tolerance test NaCl concentration result 0 + 2% + 4% + 6% + 8% + 10% +

[0082] 4. Determination of the broad-spectrum antifungal activity of JDB15 The test pathogens include 13 common pathogenic fungi including Botrytis cinerea, Fusarium graminearum, Magnaporthe grisea, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium wilt of rapeseed, Sclerotiorum rolfsii of peanut, Fusarium fusariotii, Colletotrichum syringae, Peach brown rot, Fusarium oxysporum tomato-specific type, Alternaria alternata and Microgyrus maydis, as well as two bacteria including bacterial leaf blight (PXO99) and bacterial leaf streak (XOC). Gray mold, Fusarium graminearum, rice blast, southern leaf spot of mayd, alternaria and Fusarium fujikura were deposited in the research group of Chen Xiaolin of Huazhong Agricultural University; Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium wilt of rapeseed and white rot of peanut were provided by Professor Yang Long of the College of Plant Science and Technology of Huazhong Agricultural University, peach anthracnose and peach brown rot were provided by Professor Luo Chaoxi of the College of Plant Science and Technology of Huazhong Agricultural University, bacterial leaf blight (PXO99) and bacterial leaf streak (XOC) were provided by Professor Li Guotian of the College of Plant Science and Technology of Huazhong Agricultural University, and the tomato-specific type of Fusarium oxysporum was provided by Professor Liang Wenxing of the College of Plant Medicine of Qingdao Agricultural University.

[0083] Broad-spectrum fungal antagonism: Each strain was activated on PDA medium one week in advance. Fresh strains of JDB15 were prepared one day in advance. Inoculation began in a cleanroom. Fresh mycelial cakes were punched from the edges of 5-day-old colonies of each of the 13 pathogenic fungi using a sterile 5-mm diameter punch. These cakes were then inoculated in the center of 6-cm diameter PDA dishes as target bacteria. A single colony of the activated JDB15 strain was streaked longitudinally with a sterile toothpick at two locations 1.5 cm to the left and right of the center of the cake. A blank control was inoculated with only Fusarium wilt pathogen Fusarium oxysporum. The cultures were incubated at 25°C for 2-5 days, photographed, and the transverse diameters of the pathogens in the control and treatment groups were measured. The inhibition rate (%) was calculated as: control colony diameter - treatment colony diameter / control colony diameter × 100%. Five replicates were performed for each group, with three biological replicates.

[0084] Broad-spectrum bacterial antagonism: Activate each strain in LB liquid medium two days in advance. Prepare fresh fermentation broth of strain JDB15 one day in advance. Begin inoculation in a clean bench. First, thaw a bottle of LB solid medium. Once cooled to a cool temperature, add the PX099 and XOC bacterial cultures and invert the plates. Punch wells using an Oxford cup and add varying amounts of JDB15 fermentation broth (20μl, 30μl, and 40μl). Use LB medium as the control (CK) and incubate at 30°C for two days. Photograph the plate.

[0085] Preparation of fermentation broth of strain JDB15: Inoculate a single colony of the strain into 2 mL of liquid LB medium, shake at 28°C, 180 rpm for 12 h until the culture solution becomes turbid, draw 100 μL of the culture solution and add 100 mL of liquid LB medium to continue fermentation, and shake the fermentation solution until the OD 600 =1 concentration, diluted to 1×10 7 CFU / ml, reserve for future use.

[0086] The results are as follows Figure 8 、 Figure 9 As shown in Table 3, strain JDB15 has a certain inhibitory effect on 13 plant pathogenic fungi ( Figure 8 and Table 3), has good broad-spectrum antifungal activity and significant inhibitory effect. Strain JDB15 also has significant inhibitory effects on PXO99 (bacterial leaf blight) and XOC (bacterial leaf streak) ( Figure 9 ).

[0087] Table 3 Results of broad-spectrum antifungal activity assay of JDB15 pathogens Inhibition rate Botrytis cinerea 76.35±1.62% Fusarium graminearum 64.3±1.7% Rice blast fungus 74.5±1.5% Sclerotinia sclerotiorum 65.75±2.25% Rhizoctonia solani 82.47±2.03% Peanut Sclerotium rolfsii Sacc 78.43±1.67% Fusarium fujikuroi 69.93±1.04% Fusarium wilt 71.23±1.65% Peach brown rot 72.3±2.11% Peach Anthracnose 71.73±2.17% Fusarium oxysporum tomato-specific 73.44±2.16% Bipolaris maydis 64.45±3.05% Alternaria alternata 60.45±2.18

[0088] 5. Determination of the antibacterial activity of the fermentation filtrate of strain JDB15 against banana wilt Preparation method of strain JDB15 fermentation broth filtrate: centrifuge the fermentation broth in "4" at 8000 r / min for 10 minutes, take the supernatant and filter it twice with a 22 μm sterile filter to obtain.

[0089] (1) Experimental method: Control group ( Foc 4) PDA culture medium, treatment group ( Foc 4+JDB15) Take the fermentation filtrate and add it to the cooled PDA medium at a ratio of 10% of the volume of the medium. After solidification, use a 5mm punch to punch banana wilt fungus. Foc 4. Transfer the colony to the center of the PDA and place it in a 25°C incubator. Once the control group has grown to full size, observe the growth of the treated group. Calculate the inhibition rate. Inhibition rate (%) = control colony diameter - treated colony diameter / control colony diameter × 100%.

[0090] The results showed that ( Figure 10 ), the fermentation filtrate of the strain Foc The inhibition rate of 4 was 48.85%.

[0091] (2) Experimental method: The control group (CK) was PDA culture medium, and the treatment group (JDB15) took the fermentation filtrate and added it to the cooled PDA culture medium at a ratio of 10% of the culture medium volume. After the plate was solidified, banana wilt fungi were inoculated into the center of the PDA using a 5mm punch and placed in a 25℃ incubator for culture. After two days of culture, the tip of the hyphae was inserted obliquely into a glass slide. After 24 hours, the slide was removed and observed under a microscope. The hyphae at this location were cut into small 1cm squares with a surgical blade, placed in electron microscope fixative, and subjected to gradient centrifugation using different concentrations of ethanol, critical point dried, and gold sprayed. The samples were sent to Wuhan Keruinuo Company for testing.

[0092] The experimental results are as follows Figure 11 , can be found through microscopic observation Foc Mycelial morphology of CK: Normal mycelia have a smooth surface, regular, rounded, and plump tips. Mycelia treated with JDB15 fermentation broth filtrate, however, have a rough surface, irregular swelling, and fragmented cell walls. Branches at the mycelial tips are webbed, with spherical and spindle-shaped swellings. SEM analysis revealed smooth mycelial surfaces without noticeable creases in the treated group, while mycelia in the untreated group exhibited fragmentation, distortion, and distortion.

[0093] (3) Experimental method: To determine the effect of JDB15 fermentation broth filtrate on Foc 4. The effect of spore germination will Foc 4 spore suspension (10 6 CFU / mL) and 10% of the suspension volume of sterile fermentation broth filtrate were thoroughly mixed ( Foc 4+JDB15), with an equal volume of sterile water as a control (CK). The mixture was dripped onto a hydrophobic glass slide, placed in a glass dish containing moistened filter paper, and incubated in a 28°C incubator for 12 hours. Three biological replicates were set up for each treatment. Spore germination was counted, and the spore germination rate was calculated as follows: germination rate (%) = (number of germinated spores) / total number of spores × 100%.

[0094] Experimental results ( Figure 12 ): The germination rate of the control group was about 71%, while the spore germination rate of Foc4 in the JDB15 fermentation liquid treatment group was only 22.56%, which was significantly lower than that of the control group, indicating that the JDB15 fermentation liquid filtrate can significantly inhibit the Foc 4. Spore germination.

[0095] 6. Banana seedling pot inoculation test The efficacy of the fermentation broth of strain JDB15 (prepared as described in "4") and its filtrate against banana wilt was further verified in a greenhouse at an average temperature of 28°C and a relative humidity of 70%. Banana seedlings were grown in seedling bags and transplanted into pots when they had 3 to 4 true leaves. Three treatments were set up: a negative control (H2O (sterile deionized water), a GFP- Foc 4 (positive control 1.0 × 10 7 CFU / ml), strain JDB15 (1.0 × 10 7 CFU / ml) + GFP- Foc 4 (1.0 × 10 7 CFU / ml), strain JDB15 fermentation broth + GFP- Foc 4 (1.0 × 10 7 CFU / ml) (volume ratio 1:1). First, the fermentation liquid of strain JDB15 and the filtrate of the fermentation liquid were applied to the roots of banana plants that had been damaged for soaking. The next day, 20 ml of GFP- Foc A spore suspension was applied to the roots of banana plants and 20 ml of the fermentation broth or filtrate of the JDB15 strain was added. Ten plants were used for each treatment.

[0096] According to Himaman et al. (2016), each group of banana seedlings was divided into five grades, calculated based on the percentage of chlorotic leaves in a plant: Grade 0: healthy plant, Grade 1: 1-25% chlorotic leaves, Grade 2: 26-50% chlorotic leaves, Grade 3: 51-75% chlorotic leaves, and Grade 4: more than 75% chlorotic leaves. The disease index (DI) for banana wilt is calculated as follows:

[0097] The results show that ( Figure 13 ), the disease index of banana plants treated with the fermentation liquid and fermentation liquid filtrate of strain JDB15 was significantly reduced, indicating that the fermentation liquid and fermentation liquid filtrate of strain JDB15 have a significant antagonistic effect on banana wilt and can reduce the disease index of banana plants.

[0098] 7. Evaluation of banana seedling growth promotion effect The growth-promoting effect of the antagonistic bacteria fermentation liquid on banana seedlings was further verified in a greenhouse at an average temperature of 28°C and a relative humidity of 70%. Banana seedling bags were purchased from the company and transplanted into pots. The following two treatments were set up: H2O (sterile deionized water), antagonistic strain JDB15 (1.0 × 10 7 CFU / ml). Bacterial fermentation broth was added every seven days. Treatment lasted 30 days, with 10 strains used in each treatment.

[0099] The results are as follows Figure 14 and Figure 15 The banana plants treated with strain JDB15 showed significant increases in stem diameter, plant height, leaf area, plant fresh weight, plant dry weight and root number, indicating that JDB15 can promote the growth of banana plants and has a significant growth-promoting effect.

[0100] Experimental methods: (1) Plant height: The height of the banana plant's pseudostem is measured with a 1-meter soft ruler from the part of the banana plant's pseudostem that just emerges from the soil to the intersection of the first and second unfolded leaves.

[0101] (2) Stem diameter: The diameter of the banana seedling stem is measured 2 cm above the sand surface using an electronic vernier caliper.

[0102] (3) Total leaf area: Based on the predicted leaf area of ​​“Basrsi” (MV et al., 1994) regression equation (leaf Area (m 2 ) = 0.0266 + (L*W*0.7629) (r = 0.98), where L is leaf length (m) and W is leaf width (m). The new leaf area was calculated (Zhang et al., 2008). The total new leaf area is the sum of the areas of each new leaf.

[0103] (4) Plant fresh weight: Pull out the banana from the soil with its roots, wash the soil around the banana roots with running water, and weigh the banana plant directly.

[0104] (5) Plant dry weight: Wash the bananas with running water and freeze them in liquid nitrogen for 30 min, then take them out and weigh them.

[0105] (6) Number of roots: Wash the roots with running water and count the number of thicker roots.

[0106] 8. Enzyme Activity Detection for Banana Seedlings In a greenhouse with an average temperature of 28°C and a relative humidity of 70%, we further verified the effect of the fermentation liquid of strain JDB15 on the enzyme activity of banana plants infected with Fusarium wilt. We purchased banana seedling bags from the company and transplanted them into pots. We set up the following two treatments: on day 0, we added water and fermentation liquid of strain JDB15. CK was the water treatment. On day 3, after sampling, we added Foc 4 spore liquid for infection. The results are as follows Figure 16 shown.

[0107] The results showed that the application of JDB15 fermentation broth, Foc4. Both before and after application of the spore solution, POD (peroxidase) activity and CAT (catalase) content in banana plants were significantly increased. This indicates that application of the fermentation solution of strain JDB15 not only increases POD activity and CAT content in normally growing banana plants, but also significantly enhances POD activity and CAT content in banana seedlings infected with banana wilt, significantly improving the stress resistance of banana seedlings and their resistance to banana wilt. The effect of improving stress resistance is even more pronounced in banana plants infected with banana wilt.

[0108] Experimental method: POD activity was determined using a peroxidase (POD) activity detection kit (Cat. No.: BC0090, manufacturer: Solebol), and CAT content was determined using a catalase (CAT) activity detection kit (Cat. No.: BC0205, manufacturer: Solebol).

[0109] 9. JDB15's effectiveness in controlling other pests and diseases (1) Rice blast A spore suspension of rice blast fungus was prepared with 0.05% (v / v) Tween 20 and the spore concentration was adjusted to 1×10 5 The spore solution was mixed with water at a volume ratio of 1:1 as the control group (WT), and with JDB15 at a volume ratio of 1:1 as the experimental group (WT+JDB15). The negative control was treated with water (H2O). The leaves were then sprayed onto the front of 7-day-old barley leaves, sealed with plastic wrap, and placed in the dark at 28°C for 24 hours before exposure to light. After 48 days, the plastic wrap was removed for ventilation, ensuring moisture retention. After 5 days, the inoculation results were observed and photographed. Figure 17 superior) Use scissors to cut barley leaves of appropriate size, use a 10μl gun to draw the above spore mixture and inoculate it on the front of the barley leaves. Keep in the dark at 28℃ for 24 hours and then expose to light. After 48 days, remove the plastic wrap and ventilate, pay attention to moisture retention, and observe the inoculation results after 5 days and take pictures. Figure 17 Down) The results are as follows Figure 17 As shown, the inoculation of the fermentation liquid of the strain JDB15 can significantly reduce the number and area of ​​lesions of rice blast on barley leaves compared with the control group inoculated with the spore suspension, and reduce the incidence of rice blast on barley leaves.

[0110] (2) Corn leaf spot After sporulation, the corn leaf spot fungus was washed with 0.05% (v / v) Tween20 aqueous solution to prepare a suspension, and the concentration was adjusted to 1×10 4pc / mL. Seven-day-old corn seedlings with similar growth characteristics were selected as test subjects. The spore solution was mixed with water (WT) and JDB15 (WT+JDB15) at a volume ratio of 1:1. The leaves were lightly scratched with a syringe needle, and the spore solution was applied to the scratched area with a gun. After the disease occurred, a photo was taken with a camera. The results are as follows. Figure 18 After inoculation with the fermentation broth of strain JDB15 (WT+JDB15), the lesion area of ​​corn leaf blight was significantly reduced compared with the WT group with only water added, indicating that strain JDB15 has a significant inhibitory effect on corn leaf blight.

[0111] (3) Nematodes The washed nematodes were placed in a 96-cell plate, and 100 μL of water (WT) and fermentation broth of strain JDB15 (JDB15) were added respectively. The plates were cultured at 28°C for 12 h and observed under an optical microscope. Figure 19 When JDB15 fermentation broth (JDB15) was added, the mortality rate of nematodes increased significantly, significantly higher than that of the WT group, indicating that strain JDB15 has the effect of killing nematodes.

[0112] (4) Tomato wilt The wild-type strain of Fusarium oxysporum was activated on plates for 4 days, and then the bacterial cakes were taken from the edge of each colony and shaken in PDB liquid medium. Conidia were collected for about 20 hours. Conidia were counted using a hemocytometer, and the spore concentration of tomato wilt pathogen was diluted to 1×10 7 The pathogenicity test of Fusarium oxysporum was conducted by the root dipping method. Tomato seedlings with 4 new leaves of uniform growth were selected, the soil at the roots was removed, and the roots were rinsed with clean water. The root tips were cut with scissors to create artificial wounds to facilitate the infection of pathogenic fungi. Ten tomato seedlings with uniform growth momentum were selected as a group, and their roots were immersed in a 1:1 volume ratio of (clean water + Fol ) (WT), volume ratio 1:1 (JDB15+ Fol ) (WT + JDB15), and double-distilled water (ddH2O). After soaking each group for 30 minutes, the tomato seedlings were removed and replanted in small pots filled with nutrient soil. The tomato seedlings were placed in an artificial climate chamber for cultivation. The disease status of the plants was recorded, photographed, and the disease index of the tomato seedlings was calculated. Figure 20 When the fermentation broth of strain JDB15 was added, the disease index of tomato Fusarium wilt was significantly reduced compared to the WT group. Furthermore, as the experiment progressed, the disease index of the JDB15 fermentation broth group did not significantly increase, while that of the WT group significantly increased. The disease index of the JDB15 fermentation broth group decreased even more significantly. This suggests that strain JDB15 has a significant inhibitory effect on tomato Fusarium wilt, and the inhibitory effect becomes more pronounced over time.

[0113] (5) Gray mold On a clean bench, inoculate PDA plates with a sterilized, cooled inoculation needle using an alcohol burner. The inoculation needle was then inoculated at 20°C in the dark for 3-4 days to activate the culture. 12 hours in advance, clean water and the fermentation broth of strain JDB15 were prepared and evenly sprayed onto the surface of detached tobacco leaves. A borer was sterilized using an alcohol burner on the clean bench. After cooling, a bacterial cake was punched from the edge of the colony. Fresh, healthy detached leaves, previously sprayed with clean water (WT) and fermentation broth of strain JDB15 (WT+JDB15), were inoculated with the bacterial cake. The plates were then placed on a plastic tray lined with moistened absorbent paper, sealed to maintain moisture, and incubated at 20°C in the dark. Approximately five replicates were performed for each strain and treatment. After 72 hours of incubation, the diameter of the lesions at each inoculation point was measured using the cross-hatch method. The average diameter across replicates was used to assess the pathogenicity of the strain. Figure 21 As shown in the figure, the diameter of gray mold lesions on tobacco leaves inoculated with strain JDB15 was significantly reduced, which was significantly different from that of the control group (WT).

[0114] (6) Peach Anthracnose Commercially ripe fruit (still firm but without any green skin) was rinsed with detergent and water and surface-sterilized in 1% sodium hypochlorite for 2 minutes. The fruit was then rinsed with sterile water and allowed to dry on sterile paper. A sterilized toothpick was used to pierce the fruit approximately 5 mm deep. A spore suspension of anthracnose was applied using a spreading stick, adjusting the spore concentration to 1 × 10 6 / ml, the spore liquid was mixed with clean water (WT) and the fermentation liquid of strain JDB15 (WT+JDB15) in a volume ratio of 1:1, and 10μL of the mixed conidia suspension was dropped on each wound, and 10μL of sterile water (H2O) was added to the control fruit (CK) or leaves. Each fruit has 2 inoculation sites. Three fruits were inoculated for each treatment. The inoculated fruits were placed in a plastic tray on a plastic ring with a diameter of 30mm to keep it stable. The bottom was covered with moist absorbent paper and the top was sealed with plastic film to maintain humidity. The peach fruit was cultured at 25°C for 5 days. The pathogenicity was evaluated by the diameter of the lesion. The lesion size is the average of 2 vertical diameters. The results are as shown Figure 22 When the fermentation liquid of strain JDB15 was added, the diameter of peach anthracnose lesions was significantly reduced, which was significantly different from the control group.

[0115] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A banana endophytic Bacillus Velez JDB15, characterized in that Named Bacillus velezensis , registered and deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 66431.

2. the fermented liquid of banana endophytic Bacillus Velez subtilis JDB15 or the filtered liquid of fermented liquid as claimed in claim 1.

3. A preparation, characterized in that Contains the banana endophytic Bacillus Velez JDB15 according to claim 1, or the fermentation liquid according to claim 2.

4. the application of banana endophytic Bacillus Velez JDB15 as claimed in claim 1, or fermented liquid as claimed in claim 2, or preparation as claimed in claim 3 in the preparation of antagonistic banana wilt pathogen, gray mold, Fusarium graminearum, rice blast pathogen, sclerotinia sclerotiorum, Rhizoctonia solani, rape wilt pathogen, peanut white rot pathogen, Fujikura Fusarium, peach anthracnose pathogen, peach brown rot, Fusarium oxysporum tomato special type, Alternaria alternata, corn leaf blight pathogen, bacterial leaf blight pathogen and bacterial leaf streak pathogen.

5. the application of banana endophytic Velez bacillus JDB15 as claimed in claim 1, or fermented liquid as claimed in claim 2, or preparation as claimed in claim 3 in the preparation of preventing and treating banana wilt pathogen, gray mold, Fusarium graminearum, rice blast fungus, sclerotinia sclerotiorum, Rhizoctonia solani, rape wilt pathogen, peanut white rot fungus, Fujikura Fusarium, peach anthracnose pathogen, peach brown rot, Fusarium oxysporum tomato special type, Alternaria alternata, corn leaf blight pathogen, bacterial leaf blight pathogen and / or bacterial leaf streak pathogen, or the preparation of preventing and treating nematodes.

6. the application of banana endophytic Bacillus Velez JDB15 as claimed in claim 1, or fermented liquid as claimed in claim 2 or the filtrate of fermented liquid, or preparation as claimed in claim 3 in preparing a preparation that improves banana plant resistance to banana wilt pathogen, suppresses banana wilt pathogen mycelial growth, or suppresses banana wilt pathogen spore germination.

7. Use of the banana endophytic Bacillus Velez subtilis JDB15 as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the preparation as claimed in claim 3 in promoting banana plant growth and / or improving banana plant stress resistance.

8. application as claimed in claim 7, banana endophytic Bacillus Velez subtilis JDB15 as claimed in claim 1 or fermented liquid as claimed in claim 2 or preparation as claimed in claim 3 can improve the stem thickness, plant height, leaf area, plant fresh weight, plant dry weight and / or root number of banana plants.

9. application as claimed in claim 7, banana endophytic Bacillus Velez subtilis JDB15 as claimed in claim 1 or fermented liquid as claimed in claim 2 or preparation as claimed in claim 3 can improve peroxidase activity and / or catalase content in normal banana plants and banana plants infected with banana wilt pathogen.

10. The application of banana endophytic Bacillus Velez subtilis JDB15 as claimed in claim 1, or fermented liquid as claimed in claim 2, or preparation as claimed in claim 3 in preparing a preparation that secretes protease, cellulase, amylase, pectinase, growth hormone, nitrogen fixation, potassium solution and / or chitinase activity.

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